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Endocrinology Vol. 143, No. 8 3036-3043
Copyright © 2002 by The Endocrine Society

ARTICLE

Transcript Expression of the Tuberoinfundibular Peptide (TIP)39/PTH2 Receptor System and Non-PTH1 Receptor-Mediated Tonic Effects of TIP39 and Other PTH2 Receptor Ligands in Renal Vessels

Anne Eichinger, Nathalie Fiaschi-Taesch, Thierry Massfelder, Samuel Fritsch, Mariette Barthelmebs and Jean-Jacques Helwig

Renovascular Pharmacology and Physiology, Institut National de la Santé et de la Recherche Médicale and University Louis Pasteur Medical School, Strasbourg F67085, France

Address all correspondence and requests for reprints to: Jean-Jacques Helwig, Ph.D., Pharmacologie and Physiologie Rénovasculaires, Equipe Mixte Institut National de la Santé et de la Recherche Médicale- University Louis Pasteur 0015, 11 rue Humann, Bâtiment 4, 1er étage, F67085 Strasbourg Cedex, France. E-mail: . jean-jacques.helwig{at}pharmaco-ulp.u-strasbg.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although lower than in brain, the type 2 PTH receptor (PTH2-R) has been shown to be expressed throughout the cardiovascular system. Tuberoinfundibular peptide (TIP) purified from brain is thought to be the endogenous selective ligand of the PTH2-R. In the present studies, TIP and PTH2-R mRNA expressions were evidenced by RT-PCR in rat intrarenal arteries as well as in renovascular smooth muscle cells cultured from these arteries. In the isolated perfused rat kidney (IPK), peptides known to bind to both PTH1- and PTH2-Rs, such as rat PTH (1–34) and the hybrid PTH/PTHrP peptide, [Ile5, Trp23]PTHrP (1–36), failed to exhibit improved vasodilatory effect, compared with human PTHrP (1–36), which binds only to the PTH1-R. Thus, a non-PTH1-R seemed not to be involved in the vasodilatory effects of these peptides. On the other hand, TIP exhibited complex vasoactivity, constricting the IPK at 10 nM and dilating the IPK at 1, 100, and 1000 nM. Moreover, [p-benzoyl-L-Phe4,Ile5,Trp23]PTHrP (1–36), initially described as a selective PTH2-R antagonist, also displayed a strong vasodilatory effect and therefore could not be used to check that TIP-induced vasoactivity was mediated by the PTH2-R. However, both [p-benzoyl-L-Phe4,Ile5,Trp23]PTHrP (1–36) and TIP displayed similar or even enhanced vasodilation in IPK in which PTH1-R-induced vasodilation was fully desensitized by sustained exposure to human PTHrP (1–36). Importantly, in IPK desensitized to the vasodilatory action of PTHrP (1–36), the hybrid PTH/PTHrP peptide and rat PTH (1–34), whose vasodilatory responses appeared exclusively PTH1-R dependent in naive IPK, produced a new and strong vasodilation. In conclusion, TIP and PTH2-R mRNAs are expressed in renal vessels and TIP appears as a new vasoactive peptide. Whether TIP interacts with PTH2-R could not be shown. However, these studies reveal the ability of TIP, as well as of other peptides known to bind to the PTH2-R, to dilate renal vessels in a PTH1-R-independent manner. Moreover, results obtained in IPK desensitized to the vasodilatory action of PTHrP (1–36) strongly suggest that TIP, along with PTHrP, might be coordinately involved in the regulation of renal hemodynamics.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PTHrP IS A POLYHORMONE produced throughout the normal body involved in a number of normal functions, including development, calcium transport, and smooth muscle relaxation (1, 2, 3, 4, 5, 6, 7). Among the various PTHrP species generated posttranslationally from native peptide, PTHrP (1–36) is now thought to be an authentic regulator of the development and the functions of the cardiovascular system (4, 5). Despite their limited amino acid sequence homology, PTH (1–34) and PTHrP (1–36) display comparable ability to bind and activate the type 1 PTH receptor (PTH1-R) (6, 8, 9, 10). The PTH1-R is the first identified receptor for PTH-related ligands (6). Although abundantly expressed in bone and kidney, the PTH1-R is also expressed in a number of other tissues in which it serves multiple biological roles, generally unrelated to mineral metabolism. Among them, the effects of PTHrP (1–36) on vascular tone have all been considered to be mediated by the PTH1-R. That the PTH1-R plays a significant role in blood pressure homeostasis has been convincingly documented in mice, in which targeted overexpression of PTH1-R and/or PTHrP in smooth muscle led to clear cardiovascular phenotype, including reduced blood pressure (11, 12). Furthermore, that PTH1-R plays a significant role in regional hemodynamics has notably been documented in detail in the arterial system of the kidney (4). In this organ PTH1-R not only mediates distal tubular calcium reabsorption but is also a potent dilator of the preglomerular arteriole and arteries (13, 14) as well as a stimulator of renin release (15). Evidence for intrarenal arteriolar binding sites, which equipotentially bind PTH and PTHrP, supports these findings (16). Other results have since corroborated this concept. Thus, in renal vessels of spontaneously hypertensive rats, which are widely acknowledged to have increased renal vascular resistance, PTHrP (1–36) is up-regulated, but the PTH1-R is down-regulated (17, 18). Importantly, replenishment of renovascular PTH1-R by peripheral gene delivery reduced renal tone in vitro and increased plasma renin activity, which further suggests a critical role of the PTH1-R in the regulation of systemic and renal tone (18).

PTH2-R, another member of the secretin/calcitonin family of receptors, is the second receptor identified for PTH-related ligands (19). Although sharing 51% amino acid sequence identity with the PTH1-R, the pharmacological profile of this newly discovered PTH-R differs in many ways from PTH1-R. Initial functional characterization of the human PTH2-R revealed that it is activated by PTH but not by PTHrP (19). However, the PTH2-R displays weak potency to bind PTHrP. Furthermore, in both PTH/human PTH2-R (hPTH2-R) and PTHrP/hPTH1-R interactions, residues 5 and 23 of the ligands have been proven to play critical roles for cAMP signaling and binding affinity, respectively. Indeed, replacing His5 and Phe23 in PTHrP by the corresponding PTH residues (Ile5 and Trp23, respectively), converted [Ile5, Trp23]PTHrP (1–36) into a potent activator of the PTH2-R (20). In contrast to the hPTH2-R, the rat PTH2-R (rPTH2-R) is poorly activated by PTH (21).

Although expressed less high than the PTH1-R, immunohistochemical (22) as well as in situ hybridization studies (23) revealed the hypothalamus as the primary PTH2-R expression site (24). Together with the fact that the brain has little if any PTH (25, 26), these findings prompted the search of another endogenous ligand for the PTH2-R. These studies led to the purification (27) and molecular cloning (28) of a peptide of 39 amino acids from bovine brain, the tuberoinfundibular peptide (TIP), which is structurally distantly related to PTH (28, 29, 30). TIP potently activates not only the hPTH2-R but also the rPTH2-R as well as the zebrafish PTH2-R, with EC50 values close to 1 nM (29, 30). Thus, TIP is now believed to be the primary endogenous ligand of the PTH2-R. Conversely, concentrations of TIP as high as 10 µM have been shown to be unable to stimulate cAMP production by the hPTH1-R expressed in porcine kidney cells (31). Thus, TIP functions as a selective agonist for the PTH2-R. In contrast to the numerous established roles of the PTH1-R/PTH/PTHrP system, the physiological significance of the PTH2-R/TIP system is as yet in its embryonic stages. In addition to neuronal tissues, PTH2-R has been detected in pancreas and some peripheral endocrine cells (30). PTH2-R transcript and protein have also been located throughout the cardiovascular system, including vascular smooth muscle and endothelium (22, 23), suggesting a role for the PTH2-R/TIP system in regulating vascular tone. In the kidney, these studies revealed PTH2-R expression in a few cells near the vascular pole of glomeruli, which may be part of the juxtaglomerular apparatus (22, 23, 30). The question as to whether the PTH2-R is expressed in renal vessels has, however, not been specifically addressed. The present in vitro studies aimed at providing initial physiological insight regarding the PTH2-R/TIP system in the rat renovascular system.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Collagenase, collagen, Ficoll 70, and phenylephrine were from Sigma (St. Quentin Fallavier, France). DMEM, trypsin, penicillin, streptomycin, TRIzol, RNase inhibitor, Platinum Taq DNA polymerase, molecular weight ladder, agarose, and all the primers for RT-PCR analysis were obtained from Life Technologies, Inc. (Cergy Pontoise, France). Fetal bovine serum (FBS) was purchased from BioWhittaker, Inc. (Emerinville, France). P(dT)15 primer, nucleotide mix, and Moloney murine leukemia virus reverse transcriptase came from Roche Diagnostics (Meylan, France). RQ1 RNase-free DNase was obtained from Promega Corp. (Charbonnieres, France). Restriction enzymes were purchased from Roche Molecular Biochemicals (Meylan, France). Heparin was from Sanofi Pharmaceuticals, Inc. Winthrop Industrie (Gentilly, France). Sodium pentobarbital was from Sanofi Pharmaceuticals, Inc. Sante Nutrition Animale (Libourne, France). Human [His5, Phe23]PTHrP (1–36), [Ile5, Trp23]PTHrP (1–36), rPTH (1–34) and [Bpa Ile5, Trp23]PTHrP (1–36) were synthesized by Neosystem (Strasbourg, France). Bovine TIP39 was synthesized by Sigma Genosys (Cambridge, UK). All other chemicals were of analytical or best commercial grade available.

Rat renal vessel isolation and culture of renovascular smooth muscle cells
All animal studies were performed in compliance with the French animal use rules. Two-month-old male Wistar rats weighing 200–280 g with free access to standard food and water were killed by lethal intraperitoneal injection of sodium pentobarbital. Small preglomerular arterial trees consisting mainly of arcuate and interlobular arteries (80–400 µm diameter) were isolated from excised kidneys exactly as described previously (32). Briefly, kidneys were decapsulated, longitudinally bisected, and the medulla removed. Kidney halves were pressed sequentially against stainless steel sieves of 40 and 50 mesh size and nylon sieve of 150 mesh size. The renal vascular trees, devoid of preglomerular arterioles and glomeruli, were retained on the grids and further processed for culture of renovascular smooth muscle cells (RvSMCs). All subsequent steps were performed in sterile conditions. Vascular trees were incubated for 20 min at 37 C in a 0.9% sodium chloride solution containing 0.6 mg/ml collagenase (type IA) and 1 mg/ml trypsin inhibitor, rinsed with the buffer, successively on 60 and 150 mesh grids, and then transferred in 6-well plates precoated with rat tail type I collagen (50 µg/ml) in 0.5 ml DMEM supplemented with 30% FBS, penicillin (100 U/ml) and streptomycin (0.1 mg/ml). Explants were cultured at 37 C in humidified air containing 10% CO2. When a sufficient amount of cells had grown out from the explants (typically after 10–14 d), cells were passaged by trypsinization. A homogeneous population of spindle-shaped SMCs was obtained, which can be successfully passaged more than 20 times, without noticeable changes in morphology, growth characteristics, and smooth muscle {alpha}-actin expression (32). In the present studies, RvSMCs were used at passages 4 and 5 and were cultured at 37 C in DMEM medium containing 10% FBS and antibiotics in humidified air containing 10% CO2.

RT-PCR assay for PTH1-R, PTH2-R, TIP, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
Total RNA was extracted from RvSMCs grown to 70–80% confluence as well as from isolated renal vessels, aorta, brain, kidney, and pancreas using the TRIzol method according to the manufacturer protocol. Because the coding region of TIP is within a single exon, total RNA was treated with RNase-free DNase according to the manufacturer protocol before reverse transcription (RT) of this peptide to ensure that the detected signal does not arise from contaminating genomic DNA. RT was performed with 7 µg/µl denatured total RNA using nonspecific P(dT)15 primer (2 µM) and Moloney murine leukemia virus RT at 37 C for 1 h. The concentrations of reverse transcribed RNA in PCR were adjusted in preliminary experiments to obtain similar product amplifications in the presence of 0.4 µM of the corresponding primers. The PTH1-R, PTH2-R, and TIP were detected with 1.33 µg and GAPDH with 0.63 µg reverse transcribed RNA. All reactions were done with 1.5 mM MgCl2. PCR were performed with the primers specific to rPTH1-R (33), rPTH2-R (sense: 5' GGT TGC ATT GCA CTA GG 3'; antisense: 5' GCC CAA CAG CAT TGG TC 3', and GAPDH (34). The PCR began with denaturation at 94 C for 4 min. PCR cycles were programmed as follows: 1 min at 94 C, 1 min at 58 C (PTH2-R) or 60 C (PTH1-R and GAPDH), and 1 min at 72 C. PCR was performed for 36 cycles, followed by an additional 8-min extension at 72 C. The primers and the reaction profile for TIP were adjusted to take into account the high content in guanine and cytosine in the nucleotide sequence of the gene (28). After denaturation at 95 C for 15 min, PCR cDNA was amplified by 35 cycles with denaturation at 94.5 C for 30 sec, annealing at 65 C for 45 sec, polymerization at 72 C for 30 sec, and a final extension at 72 C for 10 min (28). The primer sequences for mouse TIP detection were: 5' CTA GCT GAC GAC GCG GCC TTT CG 3' (sense) and 5'GTC CAG TAG CAA CAG CTT CTG C3' (antisense).

Amplified products were separated by electrophoresis on 1.2% (PTH1-R, PTH2-R, and GAPDH) and 2.5% (TIP) agarose gel containing 0.5 µg/ml ethidium bromide in the presence of Tris acetate EDTA buffer. PCR products were identified by their expected size of 817 bp (rPTH1-R), 540 bp (rPTH2-R), 102 bp (mouse TIP), and 415 bp (GAPDH). Control reactions were done by omitting reverse transcriptase. Agarose gels were recorded with a video system.

Restriction enzymes were used to verify the exact nature of rPTH2-R cDNA fragments. The rPTH2-R cDNA extracted from agarose was cut using BamHI and BstEII. As expected, BamHI generated three fragments of 246 bp, 196 bp, and 98 bp, and BstEII generated the two expected fragments of 138 bp and 402 bp. The exact nature of the TIP PCR product was checked by sequencing the amplified cDNA (Eurogentec, Ivoz-Ramet, Belgium). Alignment of the sequence thus obtained was introduced in a computer program named BLAST (www.ncbi.nih.gov) using the NR database. The results indicated a sequence identity close to 100%.

The isolated perfused kidney model
Male Wistar rats weighing 200–280 g were anesthetized by ip injection of sodium pentobarbital (50 mg/kg). The right kidney (0.9 ± 0.02 g, n = 67) was isolated and perfused in an open, nonrecirculating circuit as described previously (18). Briefly, the perfusion flow was adjusted during a 60-min equilibration period to achieve a pressure baseline of 80 ± 0.4 mm Hg (n = 67). Flow was kept constant thereafter. Renal vascular resistance measured at the end of the equilibration period was 8.0 ± 0.3 mm Hg/min per g-1 per ml-1 (n = 67). The perfusion medium (a Tyrode solution supplemented by 7% Ficoll 70) was maintained at 37 C, filtered continuously through a 1.2-µm sieve, and gassed with 95%O2/5% CO2. Because the isolated perfused kidney model (IPK) has little, if any, vasoconstrictor tone under basal resting conditions, vascular tone was raised after the equilibration period with sequential injections of phenylephrine (PE) for 15 sec every 2 min to induce repetitive and transient pressure peaks. The concentration of PE required to induce constrictory peaks of about 40–60 mm Hg was 6.8 ± 0.5 µM (n = 67). The tonic responses to the continuous perfusion of the various PTHrP-derived peptides, rPTH (1–34), and bTIP39 were assessed by their ability to attenuate the PE-induced pressure peaks, as indicated in the figures.

Statistics
Vasodilatory effect was analyzed by one-way ANOVA followed by Student-Newman-Keuls test to detect individual significant variations (P < 0.05). The relative tonic effects of the peptides, expressed as percent decrease or increase of the PE-induced preconstriction were compared using the t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of PTH2-R and TIP in renal vessels and RvSMCs
Renal vessels and RvSMCs were compared with aorta and brain for PTH2-R gene expression (Fig. 1AGo). The expression of the PTH2-R gene was found not only in brain but also in renal vessels and aorta as well as in RvSMCs. As a general rule, the expression of PTH2-R mRNA appeared lower than the PTH1-R expression in renal vessels and RvSMCs. Clear signal for TIP gene expression (Fig. 1BGo) was also found in renal vessels and RvSMCs as well as in aorta, brain, kidney, and pancreas.



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  		Figure 1. RT-PCR analysis of PTH1-R and PTH2-R (A) and TIP (B) gene expression in isolated intrarenal arteries and smooth muscle cells cultured from these vessels (RvSMCs) as well as in other indicated organs. Typical examples of ethidium bromide-stained gels are shown. Similar results were obtained in three to four independent tissues or cell preparations.

 
Vasoactivity of PTH1-R and/or PTH2-R ligands in the IPK
To begin to characterize a possible PTH2-R-mediated tonic effect in rat renal vessels, we decided to ask whether rPTH (1–34) or [Ile5, Trp23]PTHrP (1–36), the hybrid PTH/PTHrP peptide, would have enhanced vasoactivity in the rat IPK, compared with hPTHrP (1–36). Results are shown in Fig. 2Go. Rat PTH (1–34) and hPTHrP (1–36) exhibited similar potencies to decrease the PE-induced vasoconstriction in the rat IPK (EC50 values around 3 nM and maximum response around 60%). Moreover, the hybrid PTH/PTHrP ligand did not display improved vasodilatory potency, compared with native hPTHrP (1–36) or rPTH (1–34). Additionally, although hPTHrP (1–36) and [Ile5, Trp23]PTHrP (1–36) exhibited sustained vasodilation during peptide infusion, the dilatory response to rPTH (1–34), especially at the highest 30-nM concentration, appeared short lasted with rapid reversion over time.



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  		Figure 2. Concentration-dependent tonic effects of hPTHrP (1–36) (A), rPTH (1–34) (B), and [Ile5, Trp23]PTHrP (1–36) (C) in the isolated rat kidney perfused at constant flow. Kidney preparations were preconstricted by sequential injections of PE during 15 sec every 2 min as described in Materials and Methods. In A–C, Data points represent the pressure values of the PE-induced constrictory peaks thus generated, either in absence (none) or in presence of increasing concentrations of the various peptides, as indicated. Each concentration of the peptides has been perfused over 6-min periods. *, P < 0.05 vs. none in single-range ANOVA for repeated measures for the indicated independent renal preparations. D, The vasodilatory actions of the peptides data points represent the maximum relative decrease (percent) of the PE-induced preconstriction.

 
The full bovine sequence of TIP39 displayed complex concentration-dependent tonic effects in the rat IPK (Fig. 3Go). At 1, 100, and 1000 nM, bTIP39 dose dependently vasodilated the renal preparations preconstricted with PE. Concentration as low as 1 nM significantly dilated the IPK by approximately 10% (P = 0.035, n = 7). In contrast, at 10 nM, bTIP39 significantly increased the PE-induced preconstriction by 32.2 ± 10.1% (P = 0.046, n = 7). This vasoconstrictory response to 10 nM of bTIP39 became highly significant, compared with the level of PE-induced constriction in the presence of 1 nM bTIP39 (P < 0.001). Thus, increasing bTIP39 concentration from 1 to 10 nM augmented the PE-induced constriction by about 55%. At 100 and 1000 nM, bTIP39 reduced by approximately 20% (P = 0.041, n = 7) and approximately 40% (P < 0.001, n = 7) the vasoconstriction induced by PE.



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  		Figure 3. Concentration-dependent tonic effects of bTIP39 in the isolated rat kidney perfused at constant flow as described in Materials and Methods and Fig. 2Go. Each concentration of bTIP39 has been perfused over 6-min periods. *, P < 0.05 vs. none in single-range ANOVA for repeated measures in seven independent renal preparations. The relative vasodilatory or vasoconstrictory actions of bTIP39, expressed as percent decrease or increase, respectively, of PE-induced peaks are shown on the right axis.

 
We next chose to determine whether the vasoactivity of bTIP39 was mediated by the PTH2-R. Earlier studies of the N terminus of the PTH/PTHrP hybrid ligand [Ile5, Trp23]PTHrP (1–36) demonstrated that substitution of the photoreactive amino acid para-benzoyl-L-phenylalanine (Bpa) at position 4 selectively antagonized the PTH2-R in porcine kidney cells (35). Surprisingly, in the renovascular system described herein, [Bpa4, Ile5, Trp23]PTHrP (1–36) displayed vasodilatory effects (Fig. 4Go) at concentrations ranging from 1 nM (P = 0.044, n = 5) to 1000 nM (P < 0.001, n = 5).



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  		Figure 4. Concentration-dependent tonic effects of [Bpa4, Ile5, Trp23]PTHrP (1–36) in the isolated rat kidney perfused at constant flow as described in Materials and Methods and Fig. 2Go. Each concentration of this compound has been perfused over 6-min periods. *, P < 0.05 vs. none in single-range ANOVA for repeated measures in five independent renal preparations. The relative vasodilatory effects expressed as percent decrease of PE-induced peaks are shown on the right axis.

 
Vasoactivity of PTH2-R ligands in PTH1-R-desensitized IPK
In an attempt to selectively block the PTH1-R, we next hypothesized that, if independent from PTH1-R, the bTIP39-induced vasoactivity should be seen under conditions in which the PTH1-R undergoes profound homologous desensitization to the vasodilatory action of hPTHrP (1–36). Because PTHrP (1–36) does not activate the PTH2-R and TIP39 does not activate the PTH1-R, such an approach appeared relevant. As shown in Fig. 5AGo and reported previously (36, 37, 38, 39), continuous infusion of 200 nM hPTHrP (1–36), as expected, induced rapid, profound, and transient vasodilation of the PE-preconstricted IPK. The PE-induced preconstriction was restored, i.e. complete desensitization occurred, within 8–10 min, despite the maintenance of hPTHrP (1–36) infusion. We have shown previously (38), once desensitization to hPTHrP (1–36) has occurred in this system, vasorelaxation could not occur until hPTHrP (1–36) has been withdrawn for at least 90 min. In the present experiments, during maintenance of the infusion of 200 nM hPTHrP (1–36), desensitization was maintained for at least 16 min in control experiments. In contrast, although maintaining the infusion of the desensitizing concentration of hPTHrP (1–36), 1000 nM bTIP39 infused immediately after full PTH1-R desensitization produced a new and marked vasodilation. Thus, the vasodilation induced by bTIP39 appears to be mediated by a non-PTH1-R. Interestingly enough, the maximum vasodilation induced by 1000 nM bTIP39 in PTH1-R-desensitized renal preparations was approximately 1.6-fold greater than the vasodilation induced by the same concentration of bTIP39 in naive kidneys (63.8 ± 8.9% vs. 39 ± 4.9%, P < 0.001, n = 5) (Fig. 5Go, B and C).



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  		Figure 5. Effect of the desensitization to the vasodilatory response to 200 nM hPTHrP (1–36) on the vasodilation induced by a single concentration of 1000 nM bTIP39 in isolated kidney preparations perfused at constant flow, as described in Materials and Methods. A, Human PTHrP (1–36) at 200 nM was infused in a time-control series of IPK (open squares, n = 4) to achieve complete desensitization to the vasodilatory response to the peptide. Desensitization was kept over the experimental period. In another experimental series (open circles, n = 5), bTIP39 was infused during desensitization of hPTHrP (1–36)-induced vasodilation, as indicated. B, bTIP39 was infused in naive kidney preparations. C, Maximum vasodilatory responses to bTIP39, expressed as maximum percent decrease of PE-induced peaks in desensitized kidneys (white bar) or in naive kidneys (black bar), are shown. *, P < 0.05 vs. none in single-range ANOVA for repeated measures. #, P < 0.05 in the naive vs. desensitized comparison.

 
With these observations in mind, we then asked whether [Bpa4, Ile5, Trp23]PTHrP (1–36), known to selectively bind to the PTH2-R, retains its vasodilatory capabilities under conditions in which the PTH1-R-mediated vasodilation is fully desensitized. [Bpa4, Ile5, Trp23]PTHrP (1–36) (1000 nM), infused immediately after full PTH1-R desensitization, still produced a new and marked vasodilation (Fig. 6AGo). Thus, like bTIP39, the vasodilation induced by [Bpa4, Ile5, Trp23]PTHrP (1–36) appeared to be mediated by a non-PTH1-R. Unlike bTIP39, however, the maximum vasodilation induced by [Bpa4, Ile5 Trp23]PTHrP (1–36) in PTH1-R-desensitized renal preparations was not greater than the vasodilation induced by the same concentration of peptide in naive kidneys (35.0 ± 12.0% vs. 33.2 ± 6.0%, P < 0.05, n = 3 and 5, respectively) (Fig. 6Go, B and C).



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  		Figure 6. Effect of the desensitization to the vasodilatory response to 200 nM hPTHrP (1–36) on the vasodilation induced by a single concentration of 1000 nM [Bpa4, Ile5, Trp23]PTHrP (1–36) in isolated kidney preparations perfused at constant flow, as described in Materials and Methods. A, [Bpa4, Ile5, Trp23]PTHrP (1–36) was infused during desensitization of hPTHrP (1–36)-induced vasodilation, as indicated in Fig. 5Go (n = 3). B, [Bpa4, Ile5, Trp23]PTHrP (1–36) was infused in naive kidney preparations (n = 5). C, Maximum vasodilatory responses to [Bpa4, Ile5, Trp23]PTHrP (1–36), expressed as maximum percent decrease of PE-induced peaks in desensitized kidneys (white bar) or in naive kidneys (black bar), are shown. *, P < 0.05 vs. none in single-range ANOVA for repeated measures.

 
As shown earlier in the present study (Fig. 2Go), rPTH (1–34) as well as the hybrid PTH/PTHrP ligand had no improved vasodilatory potencies, compared with hPTHrP (1–36). Enhanced vasodilatory response to bTIP39 in PTH1-R-desensitized IPK prompted us to check whether rPTH (1–34) and the hybrid PTH/PTHrP peptide would display new vasoactivity in these kidneys. As illustrated in Fig. 7Go, A and B, under conditions in which the renal preparations were fully desensitized to the vasodilatory effect of 200 nM hPTHrP (1–36), rPTH (1–34) and [Ile5, Trp23]PTHrP (1–36) at 30 nM were indeed able to induce a new and profound vasodilation. Thus, rPTH (1–34) and the hybrid PTH/PTHrP ligand display non-PTH1-R-mediated vasoactivities when the PTH1-R-mediated vasoactivity is desensitized.



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  		Figure 7. Effect of the desensitization to the vasodilatory response to 200 nM hPTHrP (1–36) on the vasodilation induced by a single concentration of [Ile5, Trp23]PTHrP (1–36) (A) or rPTH (1–34) (B) in isolated kidney preparations *, P < 0.05 vs. none in single-range ANOVA for repeated measures in seven independent renal preparations in each series.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Evidence for the expression of the recently discovered PTH2-R in the cardiovascular system, albeit at lower levels than in brain and pancreas (22, 23, 24), raised two straightforward questions. First, are the transcripts of PTH2-R and TIP39, its most likely endogenous ligand, expressed in resistance arteries, and second, is TIP39 able to elicit PTH2-R-mediated or at least non-PTH1-R-mediated tonic effects? In an initial attempt to answer these questions, we chose the small intrarenal arteries isolated from rat kidney (60–300 µm in diameter) (32) as a vascular bed, rather than the traditionally used aorta or mesenteric arteries. This choice was motivated by the high reactivity of these vessels. The preglomerular arterial tone is well known to be regulated by sophisticated regulatory pathways, involving a number of local factors to adjust renal hemodynamics and thereby to ensure the homeostasis of the composition and the pressure of circulating blood.

TIP and PTH2-R mRNAs are expressed in renal vessels and RvSMCs
We first performed RT-PCR studies to assess the expression of both TIP and PTH2-R mRNAs in isolated renal vessels. These studies provide initial evidence for the expression of TIP and PTH2-R mRNAs in both renal vessels and RvSMCs. Because we had demonstrated earlier that the renal vascular preparations contained little, if any, tubular contamination (32) and because PTH2-R and TIP mRNAs were detected in RvSMCs cultured from isolated arteries, it appears unlikely that detection of these transcripts in renal vessels was due to tubular contamination.

In in situ hybridization studies, Usdin et al. (23) demonstrated the endothelium to be one of the sites at which PTH2-R mRNA is the most abundantly expressed. This was seen not only in large blood vessels but also within arteries in each of the organs that have been examined. These earlier studies have also demonstrated the abundant expression of PTH2-R mRNA in neural tissues (24, 29). Therefore, in the present studies, the possibility exists that part of the PTH2-R mRNA prepared from isolated arteries arose from endothelium or from perivascular nerve endings. Within the kidney, Usdin et al. (23, 30) emphasized the expression of PTH2-R in a few cells near or within the juxtaglomerular apparatus. On the other hand, in the present studies, PTH2-R mRNA was detected in isolated intrarenal arteries that have previously been shown to be devoid of glomeruli and glomerular arterioles (32). Taken as a whole, these findings strongly suggest that PTH2-R transcript is actually expressed throughout the intrarenal arterial tree. Further studies are still required to precisely define the cellular origin of PTH2-R transcript in the renal glomerulovascular network.

Rat PTH (1–34)-, [Ile5, Trp23]PTHrP (1–36)-, and hPTHrP (1–36)-induced renal vasodilations involve only the PTH1R
The present studies also aimed to provide first insight into a possible PTH2-R-mediated, or at least a non-PTH1-R- mediated, activity in renal vessels. PTHrP is a poor activator of cAMP synthesis in cells expressing either the rPTH2-R or the hPTH2-R (40). Furthermore, although activating well the hPTH2-R, rPTH (1–34) has been proven to poorly activate the rPTH2-R (21). Because PTH and PTHrP vasodilate the kidney principally through the cAMP pathway (38), PTH-induced vasodilation should not involve the PTH2-R. We indeed found no major difference in the vasodilation induced by rPTH (1–34) and hPTHrP (1–36). [Ile5, Trp23]PTHrP (1–36), the PTH/PTHrP hybrid ligand, has been proven to be a potent activator of both hPTH1-R and hPTH2-R (20). Whether [Ile5, Trp23]PTHrP (1–36) has vasodilatory potency in rat and whether this effect involves the PTH2-R has not yet been specifically documented. In the present studies, the hybrid PTH/PTHrP ligand indeed displayed potent vasodilatory capabilities in the rat IPK but was unable to display improved vasodilatory activity, compared with hPTHrP (1–36). Thus, rPTH (1–34)-, [Ile5, Trp23]PTHrP (1–36)- and hPTHrP (1–36)-induced renal vasodilation, presumably do not involve non-PTH1 receptors.

TIP displays vasoactive properties
Unlike PTH, TIP39 is acknowledged as a potent and specific activator of the PTH2-R in all species tested to date (30, 40). TIP39 appeared, therefore, to be the most likely selective endogenous agonist of the PTH2-R. Together with the presence of TIP39 mRNA in renal vessels reported herein, these findings raise the question of the vasoactivity of bTIP39. Although a significant vasodilatory effect of bTIP39 was seen at a concentration as low as 1 nM, higher concentrations of bTIP39 exhibited biphasic tonic effects: a vasoconstrictory effect at 10 nM that became vasodilatory again at 100 and 1000 nM. The strong vasodilatory effect of bTIP39 at 1000 nM was confirmed in a different series of experiments using the IPK (Fig. 5Go). The mechanism by which nanomolar concentrations of bTIP39 enhanced the PE-induced preconstriction in the IPK is unclear. TIP39 has been shown, albeit poorly, to retain some potency for binding to the PTH1-R (31). Thus, TIP39 may function as a weak antagonist of the PTH1-R. It has been shown to inhibit rPTH (1–34)- or hPTHrP (1–36)-induced cAMP in PTH1-R expressing cells with median inhibitory concentration values in the micromolar range (31). Therefore, the possibility that the vasoconstrictory effect of bTIP39 seen herein was due to a predominantly antagonistic action of 10 nM bTIP39 on endogenous PTHrP-induced vasodilation should not be ruled out. Alternatively, activation of multiple receptors with different EC50 inducing multiple signaling pathways is also possible. Most importantly, what the present studies clearly demonstrate is not only that PTH2-R and TIP39 mRNAs are expressed in intrarenal arteries but also that bTIP39 is endowed with a complex vasoactivity. This finding raises the possibility that TIP39 might be a new autocrine/paracrine peptide involved in the regulation of renal hemodynamics.

Is the vasoactive response to bTIP39 mediated by the PTH2-R?
In competition-binding studies (35), in porcine kidney cells stably expressing either the hPTH1-R or the hPTH2-R, the photoreactive analog [Bpa4, Ile5, Trp23]PTHrP (1–36) has been proven to bind to, but not activate, the PTH2-R with high affinity but did not bind at all to the PTH1-R (median inhibitory concentration > 1000 nM). Thus, in these studies [Bpa4, Ile, Trp23]PTHrP (1–36) appeared as a promising antagonist of the hPTH2-R. We therefore decided to use this peptide to demonstrate that the renal vasodilatory response to bTIP39 was mediated by the PTH2-R. Unfortunately, however, this compound displayed unexpected vasodilatory potency in the rat IPK, precluding its use as a possible selective antagonist of the renal vascular rPTH2-R. The archetypal PTH1-R antagonist [Nle8,18, Tyr34]bPTH (3–34) could not be used either because it has been shown, in COS-7 cells stably expressing both receptors subtypes, to cause a significant activation of the hPTH2-R under conditions in which no effect on the hPTH1-R was observed (41). On the other hand, deletion of two, six, or eight residues from the N terminus of TIP39 dramatically improved apparent binding affinities of the truncated peptides to the hPTH1-R but still was unable to activate the human receptor (28, 31, 42). TIP (7–39) and TIP (9–39) thus appeared as powerful antagonists of the hPTH1-R, with higher binding affinity than previously described hPTH1-R antagonists, such as [Nle8,18, Tyr34]bPTH (3–34), [D-Trp12, Tyr34]bPTH (7–34) or PTHrP (7–34) (28, 31, 42). However, although greater than the PTH1-R antagonists, the PTH1-R/PTH2-R selectivity of TIP (7–39) and TIP (9–39) clearly appears insufficient to discriminate with certainty between the two receptors. In support of this, in human embryo kidney 293 cells stably expressing either the hPTH1-R or the hPTH2-R, 100 nM TIP (7–39) displaced 100% of bound labeled PTH from PTH1-R but also 70% of bound labeled TIP39 from PTH2-R (42), thereby precluding its use as a selective antagonist of the PTH1-R. Thus, neither TIP (7–39) nor TIP (9–39) appears as ideal tools to selectively block the PTH1-R in the rat. Incidentally, how TIP (7–39) and TIP (9–39) bind to the rat PTH receptors as they do for the human receptor is not yet known. Thus, the nature of the PTH-R that mediated TIP39- and [Bpa4, Ile5, Trp23]PTHrP (1–36)- induced dilations in rat renal vessels could not be clarified in the present studies. Additional studies using mouse genetic models of selective vascular deletion of PTH1-R or PTH2-R and receptor-specific antagonists, as they become available, should shed further light on the pharmacology of PTHrP and TIP and their receptors in the vascular wall.

TIP, PTH, PTH/PTHrP hybrid peptide, and Bpa4-substituted peptide induce vasodilation during PTH1-R desensitization
A number of authors have shown that PTH1-R-mediated vasodilation undergoes homologous desensitization in response to short-term exposure to PTHrP (1–36) or PTH (1–34) in various vascular models (36, 37, 38, 39), including the rat and the rabbit IPK (38, 43). Using this kind of approach, we now report that both [Bpa4, Ile5, Trp23]PTHrP (1–36) and bTIP39 were still able to vasodilate the kidney when the PTH1-R-mediated vasodilation was profoundly desensitized by uninterrupted exposure to PTHrP (1–36). PTH1-R homologous desensitization also unmasked new potent vasodilatory responses to rPTH (1–34) and the PTH/PTHrP hybrid ligand in the PTH1-R-desensitized IPK. Thus, PTH and the PTH/PTHrP hybrid ligand, whose vasodilatory effects were mediated only by the PTH1-R in naive kidneys, became able to interact with another unknown non-PTH1-R when renal vessels were fully desensitized to the vasodilatory effect of 200 nM hPTHrP (1–36). Additionally, PTH1-R desensitization, differently modulated TIP-, PTH-, hPTH/PTHrP peptide-, and Bpa4-substituted peptide vasodilations: It unmasked non-PTH1-R-mediated vasoactivity in response to rPTH (1–34) and [Ile5, Trp23]PTHrP (1–36), increased bTIP39-induced vasodilation, and had no effect on the vasodilation induced by [Bpa4, Ile5, Trp23]PTHrP (1–36). Alternatively, the possibility that these peptides are acting on multiple PTH receptors with different EC50 values should not be ruled out. Overall, these findings, strongly suggest that [Bpa4, Ile5, Trp23]PTHrP (1–36) and bTIP39 possess agonistic dilatory properties in renal vessels. Although unable to demonstrate the involvement of the PTH2-R, these findings at least reveal that the four PTH2-R ligands tested herein are able to vasodilate the rat IPK by interaction with a receptor that is not the PTH1-R. The known PTH1-R/PTH2-R-binding selectivity of these peptides in human (6, 21, 30) only suggests that the PTH2-R might be involved. Further investigations are required to answer this crucial question. Finally, the fact that the PTH1-R requires to be desensitized to reveal or modulate non-PTH1-R-mediated vasoactivities indicates that activation of the PTH1-R somehow modulates the activity of this second non-PTH1-R. Whether this unknown PTH-R is the PTH2-R and whether there is a cross-talk between the PTH2-R and the PTH1-R in rat is an open question.

In summary, the present results demonstrate that: 1) TIP39 and PTH2-R mRNAs are expressed in renal vessels; 2) rPTH (1–34) and the PTH/PTHrP hybrid peptide, [Ile5, Trp23]PTHrP (1–36) fail to produce increased vasodilatory responses, compared with hPTHrP (1–36); 3) bTIP39 possesses complex vasoactive properties in renal vessels; 4) [Bpa4, Ile5, Trp23]PTHrP (1–36), which has initially been described as a selective PTH2-R antagonist, exhibits dilatory properties in renal vessels; and finally 5) the renal vascular responses to bTIP39, rPTH (1–34), [Ile5, Trp23]PTHrP (1–36) and [Bpa4, Ile5, Trp23]PTHrP (1–36) are conserved or even enhanced under conditions in which the PTH1-R-mediated vasodilation is fully desensitized by hPTHrP (1–36). In conclusion, these findings suggest that TIP39, acting via a PTH-R different from the PTH1-R, may participate in the functional regulation of renovascular blood flow. Moreover, they suggest the existence of a cross-talk between PTH1-R and this unknown PTH-R and that TIP, along with PTHrP, might be coordinately involved in the regulation of renal hemodynamics.


   Acknowledgments
 
The authors warmly thank Mrs. Sylvie Rothhut and Suzanne Wendling for skilled technical assistance and Mr. Alain Lambert, research engineer, for designing and building devices of the isolated perfused kidney settings.


   Footnotes
 
This work was supported by the French National Institute of Health and Medical Research and the University Louis Pasteur, Strasbourg, France (Equipe Mixte Institut National de la Santé et de la Recherche Médicale-ULP 0015).

This work is part of the thesis of A.E.

Abbreviations: Bpa, Para-benzoyl-L-phenylalanine; FBS, fetal bovine serum; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; hPTH2-R, human PTH2-R; IPK, isolated perfused kidney; PE, phenylephrine; PTH1-R, type 1 PTH receptor; PTH2-R, type 2 PTH receptor; RT, reverse transcription; RvSMC, renovascular smooth muscle cell; TIP, tuberoinfundibular peptide.

Received December 10, 2001.

Accepted for publication April 18, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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