Tuberoinfundibular Peptide of 39 Residues: A New Mediator of Cardiac Function via Nitric Oxide Production in the Rat Heart
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内分泌学杂志 2005年第5期
Institute of Physiology (G.R., P.E., Y.A., K.D.S.) and Institute of Anatomy (W.K.), Justus-Liebig-University, D-35392 Giessen, Germany
Address all correspondence and requests for reprints to: Dr. Günter Ross, Institute of Physiology, University of Giessen, Aulweg 129, D-35392 Giessen, Germany. E-mail: guenter.ross@physiologie.med.uni-giessen.de.
Abstract
Tuberoinfundibular peptide (TIP39) was initially identified as a neurotransmitter and agonist of the PTH2 receptor, which is expressed in the cardiovascular system. This study documents for the first time the cardiac expression and function of TIP39.
Expression was analyzed via RT-PCR. Function was characterized on Langendorff-perfused rat hearts as left ventricular developed pressure (LVDP) and on isolated cells via a cell edge detection system. cGMP levels were detected with RIA. Tuberoinfundibular peptide (TIP39) mRNA was found to be constitutively expressed in coronary endothelium cells, isolated cardiomyocytes, ventricles, atria, and aorta. At first we investigated the vasodilatory properties of TIP39 (100 nM) in the presence of L-nitro-arginine (L-NA, 100 μM). Surprisingly, TIP39 had no vasodilatory effect but decreased LVDP by 35 ± 7%. In the absence of L-NA, addition of TIP39 decreased LVDP by 8 ± 2%. The PTH2 receptor antagonist Trp23-Tyr36-PTHrP (1–36, 100 nM) abolished this TIP39 effect in the presence of L-NA. The experiments with isolated cardiomyocytes provided similar results. TIP39 (10 nM) lowered the contraction amplitude by 6 ± 3%. In the presence of L-NA (100 μmol/liter), TIP39 lowered the amplitude by 34 ± 6%. cGMP concentration in cardiomyocytes was stimulated by TIP39 (10 nM) in the same range as by the nitric oxide (NO) donor SNAP (100 μM). In the presence of L-NA, this increase was abolished. These results suggest that an inhibition of endogenous NO production unmasks a profound negative inotropic effect of TIP39 that is mediated by an activation of the PTH2 receptor. The results obtained with isolated cardiomyocytes suggest that myocyte-derived NO, rather than vascular NO, is responsible for this effect. cGMP seems to be the downstream signal of produced NO.
Introduction
CURRENTLY DIFFERENT PTH receptors in mammals are known, namely the PTH1 receptor (PTH1r) and the PTH2 receptor (PTH2r). The expression of both receptors has been described in the cardiovascular system (1, 2, 3). These receptors are members of the same subgroup of the superfamily of heptahelical G protein-coupled receptors (4). The PTH2r has a homology of 51% to the PTH1r in its amino acid sequence (2).
The natural ligands of the PTH1r are PTHrP and PTH. Whereas PTH acts as a systemic mediator, which is synthesized from the parathyroid glands, PTHrP developed its properties in a paracrine or intracrine manner. PTH is known as a regulator of calcium homeostasis. PTHrP was first mentioned in combination with the clinical diagnosis of humoral hypercalcemia of malignancy. This syndrome is a common complication of lung and certain other cancers (5). Recently PTHrP was also identified as a factor with local cell regulatory properties. PTHrP can be released from these tissues under various physiological and pathophysiological conditions. In the cardiovascular system, for instance, PTHrP modulates heart rate, contraction, and vascular tonus (6). These effects have been linked to the PTH1r.
The PTH2r discriminates between PTH and PTHrP. Previous studies had described activation only by PTH (7, 8). However, Usdin et al. (9) isolated another peptide from the bovine hypothalamus that binds and activates this receptor. This peptide consists of 39 residues and has been designated tuberoinfundibular peptide (TIP39). A comparison among the sequences of TIP39, PTH, and PTHrP suggests that these peptides are all relatives (10).
Furthermore, phylogenetic analyses suggest that TIP39 is the archetype from which PTH and PTHrP was developed (11). Although PTH binds and activates the PTH2r, TIP39 seems to be the natural ligand of this receptor because PTH binds only with a low affinity to rat PTH2r, weakly stimulates accumulation of cAMP, and has no influence on intracellular calcium (12, 13). Another argument for this assumption is that the PTH2r is highly expressed in pancreas and brain, although the brain is a compartment with no detectable levels of PTH (3). Various tissues of human, e.g. brain, spinal cord, kidney, or heart (14), and rat, e.g. aorta, brain, renal vessels, kidney, and pancreas (15), express TIP39 mRNA, but its physiological role is presently unclear. In hypothalamus preparations an addition of TIP39 (100 nM) increased the release of many hypothalamic-releasing factors, e.g. CRH (16). Furthermore, TIP39 is released from supraspinal fibers that are involved in nociception. Peripherally injected TIP39 was able to stimulate nociceptors (17). It seems that TIP39 acts as not only a neuropeptide but also a vasoactive substance in renal vessels (15). The vasoactive properties are concentration dependent. A concentration of 1 nM results in vasodilatation, 10 nM leads to vasoconstriction, and 100 nM results in vasodilatation again.
Because the effects of the other members of the PTH family in cardiomyocytes are well documented, it is of great interest to know whether TIP39, the new member of this family, has any properties in this system and whether it is expressed in cardiac cells.
Furthermore, the question of whether an activation of the PTH2r by TIP39 on cardiac cells activates the same pathways described in the literature for other cells must be answered. The aim of this study was to investigate these open questions.
Materials and Methods
Experimental animals
Wistar rats weighting 200–250 g were used in the experiments. All animal studies were performed in accordance with guidelines described in the National Institutes of Health Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (publication no. 85–23, revised 1996). The animals were kept under standardized conditions of temperature, humidity, and light. They had free access to standard diet and drinking water ad libitum.
Cell culture
Cardiomyocytes (CMCs) and coronary endothelial cells (CECs) were obtained from rats (18). CECs were cultured as previously described (19, 20). Briefly, hearts were perfused with collagenase; the ventricles were chopped and dissolved in suspension. This suspension contained CMCs as well as CECs. To separate the cell types, the suspension was centrifuged. The supernatant contained the CECs, whereas the CMCs were in the sediment. To obtain CMCs, the sediment was resuspended, the cells were plated, and in a following step cultured at 37 C. After 2 h, cells were harvested. These cells were subsequently used for RNA extraction or cell contraction experiments. To obtain CECs, the supernatant was trypsinized and plated at a density of 106 cells on 100-mm plastic dishes. The cells were cultured at 37 C in medium 199 with Earle’s salt supplemented with penicillin G (100 IU/ml), streptomycin (100 μg/ml), and fetal bovine serum (10% vol/vol). The medium was renewed every second day.
After 5 d, when the cells had grown to confluence, they were trypsinized in PBS. Afterward the cells were prepared and used for RNA extraction. The purity of these cultures was more than 95% endothelial cells. The remaining cells were contaminations with smooth muscle cells, pericytes, and fibrocytes. The growth of other nonendothelial cells was inhibited by trypsinization and the very rapid attachment and growth of endothelial cells on the culture dishes.
PCR
Total RNA was extracted from CMCs and CECs as well as kidney, ganglion stellatum, aorta, left and right ventricle, and atria using the TRIzol method according to the manufacturer’s protocol. To avoid genomic contamination, RNA was treated with RNase-free DNase according to the manufacturer’s protocol. Treatment was performed for 1 h at 32 C in a final volume of 50 μl, containing 10 μl RNA, 5.0 mM MgCl2 (Invitrogen Life Technologies, Carlsbad, CA), 40 U/μl rRNAsin (Promega, Mannheim, Germany), and 1 U/μl RNase-free DNase (Novagen, Madison, WI). Thereafter DNA was precipitated by phenol-chloroform. Reverse transcriptase reactions were performed for 1 h at 37 C in a final volume of 5 μl using 2 μg RNA, 100 ng oligo (dT; Roche Molecular Biochemicals, Mannheim, Germany), 8 U/μl rRNAsin (Promega), and 60 U/μl Moloney murine leukemia virus reverse transcriptase (Invitrogen Life Technologies). Aliquots (1.5 μl) of synthesized cDNA were used for PCR in a final volume of 10 μl containing 0.3 mM of primer pairs (Invitrogen Life Technologies), 0.4 mM deoxynucleotide triphosphates, 1.5 mM MgCl2, and 5 U/μl Taq-DNA-polymerase (Invitrogen Life Technologies).
The quality of cDNA was tested with hypoxanthine-guanine-phosphoribosyl-transferase (HPRT) as a housekeeping gene. The following cycle conditions were used for HPRT: after denaturation for 3 min at 94 C, PCR cDNA was amplified by 25 cycles with denaturation at 94 C for 30 sec, annealing at 63 C for 45 sec, polymerization at 72 C for 30 sec and a final extension at 72 C for 3 min. To detect the expression of TIP39, we used two different primer pairs that were previously described by Eichinger et al. (15) [primer I-sequences: 5'-CTA GCT GAC GAC GCG GCC TTT CG-3' (sense); 5'-GTC CAG TAG CAA CAG CTT CTG C-3' (antisense)] and Della Penna et al. (13) [primer II-sequences: 5'-CTT GGG TAG CCC CCT GTC TCG G-3' (sense); 5' GGG CGC GTC CAG TAG CAA CAG C-3' (antisense)]. For primer I we used the following cycle conditions: after denaturation for 3 min at 94 C, PCR cDNA was amplified by 35 cycles with denaturation at 94 C for 30 sec, annealing at 62 C for 30 sec, polymerization at 72 C for 30 sec, and a final extension at 72 C for 3 min. For primer II we used the following cycle conditions: after denaturation for 3 min at 94 C, PCR cDNA was amplified by 38 cycles with denaturation at 94 C for 30 sec, annealing at 67 C for 30 sec, polymerization at 72 C for 30 sec, and a final extension at 72 C for 3 min. Amplified products were separated by electrophoresis on 2% agarose gel containing 0.04 μl ethidium bromide in Tris-acetate EDTA buffer. PCR products were identified by their expected size of 131 bp (HPRT), 102 bp (TIP39, primer I), and 157 bp (TIP39, primer II). Agarose gels were recorded with a video system.
Heart perfusion
In vitro experiments were performed in isolated, saline-perfused rat hearts as described previously (21). The hearts were mounted on a Langendorff perfusion system in a temperature-controlled chamber (37 C). The chamber was filled with humidified air during perfusion of hearts at 37 C and constant pressure (50 mm Hg) for an initial stabilizing period of 10 min. Diastolic pressure was adjusted to 12 mm Hg. After the period of stabilization, the perfusion was switched to constant flow. Hearts were perfused with oxygenated saline medium [composition of the perfusate (millimoles per liter): 124.0 NaCl, 2.7 KCl, 0.4 NaH2PO4, 1.0 MgCl2, 1.8 CaCl2, 24.0 NaHCO3, and 5.0 glucose, gassed with Carbogen (95% O2 + 5%CO2) (pH 7.40]. During administration of the different substances, hearts were perfused according to the protocols specified below in Results.
During the experiments, the following parameters were monitored: heart rate, systolic pressure, diastolic pressure, left ventricular developed pressure (LVDP, defined as left ventricular end-systolic pressure minus left ventricular end-diastolic pressure), and aortic pressure. In each group, six hearts were investigated.
Single-cell contraction
Ventricular cardiomyocytes were isolated from male rat hearts and paced at a constant frequency of 2 Hz as described before (18, 22). Each preparation of ventricular cardiomyocytes used in this study contained a mixed population of cells from two rat heats to reduce the influence of intraindividual differences. In each of these preparations, basal contractile responsiveness was determined from three cells. Cells selected for analysis did not beat spontaneously.
On average, less than 15% of all cells showed spontaneous beating within 60 sec. From the remaining cells, those cells that beat regularly for 60 sec if they were paced with 1 Hz were selected. Cell shortening was monitored using a cell-edge detection system from which the following parameters were calculated using the software MUCEL (Heidelberg, Germany): cell shortening is expressed as percentage of systolic cell length relative to diastolic cell length (L/L). The kinetics of contraction are expressed as rate constants for maximal contraction (Cmax) and relaxation velocity (Rmax). They were calculated by normalizing maximal contraction and relaxation velocities to peak contraction. In each case, cells were constantly paced and five contractions were recorded every 15 sec. The average of these recordings was used as one data point. The measurements on individual cells were repeated four times, and the average of these four measurements was used as the average contractile performance of individual cells. From each preparation, three cells were analyzed for each condition, and the same experiments were repeated with other preparations.
cGMP
CMCs were isolated from rats as mentioned above. The cultured CMCs were stimulated with different agents and then disintegrated with ethyl alcohol (100%). The lysate was centrifuged (12,000 rpm, 2 min). The supernatant was used for cGMP determination and the pellet for analysis of protein content. cGMP was determined by RIA using the cGMP [3H] assay system (code TRK500, Amersham Biosciences, Freiburg, Germany) according to the manufacturer’s protocol (23). The protein content was measured as previously described (20). The cGMP concentration was normalized to cellular protein content.
Agents used
The following agents were used: PTHrP 1–34 (100 nM; Bachem, Torrance, CA); TIP39 (0.1, 10, 50, 100, 200 nM; Bachem); N-nitro-L-arginine (L-NA) (100 μM; Sigma-Aldrich, Munich, Germany); Trp23-Tyr36-PTHrP 1–36 (PTH2r-antagonist) (24) (100 nM; Bachem, Torrance, CA); PTHrP (7–34) (100 nM; Bachem); and S-nitoso-N-acetylpenicillamine (SNAP) (100 μM; Calbiochem, Darmstadt, Germany).
Statistics
Quantitative results are expressed as means ± SEM. In experiments with more than two groups, ANOVA was used for comparison with a Student-Newmann-Keuls test for post hoc analysis. In cases in which two groups were compared, conventional t tests were performed. P < 0.05 was used as a significant difference between groups.
Results
Expression of TIP39
Expression of TIP39 was investigated in ganglion stellatum, left ventricle, right ventricle, atria, CECs, aorta, kidney, and CMCs (Fig. 1). To ensure amplification of TIP39, we used two different primer pairs. The primer pair one (pair 1) amplified a 102-bp fragment and the pair two (pair 2) a 157-bp fragment. The HPRT primers amplified a product of 131 bp. In all tissues or cells mentioned, we demonstrated an expression of TIP39 by both primers.
FIG. 1. TIP39 expression in cardiovascular tissues and cells. Typical examples of ethidium bromide gels are shown. RT-PCR analysis of HPRT (housekeeping gene) and TIP39 gene expression in ganglion stellatum (GS), left ventricle (LV), right ventricle (RV), atrium (AT), CECs, aorta (A), kidney (K), and CMCs. In every gel, a negative control (C) was made. This negative control contained all PCR substances without RNA sample. Similar results were obtained in three to four independent tissues or cell preparations.
A negative control was included on each gel (all PCR components without RNA sample). These controls were always negative. With both primers, we observed a stronger expression of TIP39 in the left ventricle and kidney. The expression in other tissues and cells were weaker.
Influence of TIP39 on contraction of isolated hearts
We wanted to first investigate the ability of TIP39 to vasodilate coronary vessels. Therefore, the coronary vessels were preconstricted with L-NA (100 μM) for 20 min. As control we used PTHrP, a PTH1r agonist. An addition of PTHrP decreased aortic pressure within a few minutes (Fig. 2). However, TIP39 (100 nM) showed no influence on perfusion pressure (Fig. 2). Surprisingly, under these conditions, TIP39 decreased LVDP about 35% (from 109.9 ± 9.9 to 71.8 ± 6.8 mm Hg; n = 6; P < 0.05; Fig. 3). No decrease of LVDP was observed with PTHrP (data not shown).
FIG. 2. Influence of PTHrP or TIP39 on the aortic pressure (AP). After a period of stabilization (10 min), coronary vessels and the aorta were preconstricted by L-NA (100 μM; 20 min). PTHrP (100 nM) showed a complete vasodilation within 10 min. TIP39 (100 nM) had no influence on aortic pressure. *, P < 0.05 (PTHrP: pretreatment vs. posttreatment, n = 6 hearts in each group). The relative vasodilative effect was expressed as percent decreases of AP before treatment. Perfusion pressure was initially set to 50 mm Hg.
FIG. 3. Influence of TIP39 (100 nM) on LVDP in presence of L-NA. After a period of stabilization (10 min), hearts were perfused with L-NA for 20 min followed by treatment with TIP39 (see Fig. 2). *, P < 0.05 (TIP39 vs. control, n = 6 hearts in each group).
Next we determined the ability TIP39 (100 nM) to lower LVDP under basal conditions without pretreatment with L-NA. In the absence of L-NA, TIP39 decreased LVDP of isolated, saline-perfused rat hearts about 8% (from 103.8 ± 8.0 to 91.4 ± 3.6 mm Hg; n = 6; P < 0.05). This decrease reached a maximum after 5 min (Fig. 4).
FIG. 4. Influence of TIP39 (100 nM) on LVDP in absence of L-NA. After a period of stabilization (10 min), an addition of TIP39 decreased LVDP. *, Difference between the control and TIP39 group after the beginning of treatment (P < 0.05, n = 6 hearts in each group).
In the following experiments, we investigated different concentrations of TIP39 with regard to LVDP. Therefore, we performed experiments with and without pretreatment of L-NA.
We used concentrations from 0.1 to 100 nM TIP39. Without L-NA (Fig. 5), a concentration of 100 nM TIP39 decreased LVDP 8.1 ± 2.2% (P < 0.05). At a concentration of 50 nM, TIP39 developed only a very weak effect (decrease of 4.0 ± 3.3%). Lower concentrations of TIP39 had no effects. The concentration-response curve was repeated in the presence of L-NA (Fig. 5). A concentration of 50 nM TIP39 caused a significant decrease of LVDP (16.3 ± 0.8%, P < 0.05). An addition of 100 nM showed a maximum decrease of LVDP (34.7 ± 6.8%, P < 0.05). All subsequent experiments were performed at a concentration of 100 nM.
FIG. 5. Concentration-response relationship for TIP39 in the presence and absence of L-NA (100 μM) in isolated perfused rat hearts. In the first group (, Tip39 (–) L-NA) treatment of TIP39 (5 min) preceded a period of stabilization (10 min). In the second group (, TIP39 (+) L-NA), the stabilization period (10 min) followed the L-NA treatment for 20 min and after this, hearts were treated with TIP39 at different concentrations (0.1,10, 50, and 100 nM) in combination with L-NA. *, #, P < 0.05 vs. initial situation before TIP39 treatment (n = 6 hearts for each concentration). The relative inotropic effects are expressed as percent decrease of LVDP before TIP39 treatment.
The strong decrease of LVDP caused by TIP39 in the presence of L-NA was attenuated by cotreatment with both receptor antagonists (PTH2r-antagonist: Trp23-Tyr36-PTHrP(1–36), PTH1r-antagonist PTHrP(7–34) [124.0 ± 4.7 (before treatment) to 124.5 ± 3.0 mm Hg (after treatment); n = 6; P > 0.05; Fig. 6]. The peptide Trp23-Tyr36-PTHrP binds with a high affinity to the PTH2r without activating this receptor (24). Because this peptide also activates the PTH1r, we used the PTH1r-antagonist PTHrP(7–34) (25). This peptide binds to this receptor, but the activating domain for the cAMP pathway is lacking. Both peptides were perfused in the presence of L-NA for 5 min before and during treatment with TIP39.
FIG. 6. Presence of Trp23-Tyr36-PTHrP(1–36; PTH2r-antagonist, 100 nM) abolished the influence of TIP39 (100 nM; 10 min) on LVDP in the presence of L-NA and the PTH1r antagonist PTHrP(7–34). Perfusions were performed with a period of stabilization (10 min), after a period of treatment with L-NA (20 min) in the presence of both receptor antagonists [RA; Trp23-Tyr36-PTHrP)(1–36) and PTHrP(7–34)] followed by a treatment of TIP39 in the presence of above-mentioned substances.
Influence of TIP39 on contraction amplitude of isolated CMCs
In a subsequent experimental step, we investigated whether the influence of TIP39 on the contraction is a direct effect on CMCs. Therefore, we investigated a concentration-response relationship on the single cell level. Experiments were performed with and without L-NA. The contraction amplitude of cell shortening (L/L) was normalized to diastolic cell length (percent). The concentrations tested were 0.1–10 nM TIP39 (Fig. 7A).
FIG. 7. A and B, Concentration-response relationship for TIP39 in the presence and absence of L-NA (100 μM) in single-cell preparations (CMC) on L/L normalized to L/L of controls. The CMC was stimulated at 2 Hz. In the first group [, Tip39 (–) L-NA], cells were treated only with TIP39 [0.1 nM (n = 90), 1 nM (n = 63), and 10 nM (n = 63)]. In the second group [, TIP39 (+) L-NA], the treatment of cells with TIP39 preceded an incubation period of 20 min with L-NA. The cells were treated with different concentrations of TIP39 [0.1 nM (n = 63), 1 nM (n = 42), and 10 nM (n = 30)]. *, #, P < 0.05 vs. untreated [0.1 (n = 87), 1 (n = 66), and 10 nM (n = 54)] cells or cells treated only with L-NA [0.1 (n = 66), 1 (n = 39), and 10 nM (n = 26)], respectively (A). Comparison of the influence of TIP39 (10 nM) (n = 63) alone and in the presence of L-NA (100 μM) (n = 30) on L/L, Cmax, and Rmax. *, P < 0.05 vs. untreated (n = 54) cells or cells treated only with L-NA (n = 26), respectively. The relative effects expressed as percent decrease of L/L, Cmax, and Rmax of treated (TIP39 ± L-NA) vs. untreated (± L-NA) cells (B).
Without L-NA, only a small decrease of L/L of 13.5 ± 3.5% (P < 0.05) was observed at 1 nM TIP39. An even higher concentration showed only a trend (decrease of 6.2 ± 2.7%; P = 0.3). In the presence of L-NA, a concentration of 1 nM TIP39 showed a decrease of 24.4 ± 4.7% (P < 0.05). At a concentration of 10 nM TIP39, a maximum decrease of the contraction amplitude was achieved (34.0 ± 5.9%; P < 0.05).
This negative contractile effect went along with reduced maximal relaxation (Rmax, decrease: 19.8 ± 8.9%) and contraction velocities (Cmax, decrease: 16.4 ± 5.3%, Fig. 7B). In all subsequent single-cell experiments, a concentration of 10 nM TIP39 was used.
Figure 8, A and B show representative single-cell contractions of individual rat ventricular cardiomyocytes paced at 2 Hz in the different groups. The figures describe the change in cell length normalized to diastolic cell length. Figure 8A shows a contraction in the control () and TIP39 groups . TIP39 reduced the contraction amplitude in comparison with the control. Figure 8B shows the same results between the L-NA control () and TIP39+L-NA () groups.
FIG. 8. A and B, Representative single-cell recordings of the time course of the length of individual rat ventricular CMCs paced at 2 Hz in the different groups. L/L is expressed as percentage of systolic cell length relative to diastolic cell length. , Control; , TIP39 (A); , control (L-NA); , TIP39 (+) L-NA (B).
Detection of cGMP
cGMP is a well-known downstream target in the nitric oxide (NO) signaling cascade. Because our results suggest an interaction of the PTH2r with the NO cascade, we wanted to know whether the concentration of cGMP is increased in cardiomyocytes in the presence of TIP39 (Fig. 9).
FIG. 9. cGMP concentrations in response to TIP39 were normalized to the protein content of each dish of different preparations. Cells were treated with TIP39 (10 nM) alone and in the presence of L-NA (100 μM) with a preincubation period of 20 min. As positive control, cells were treated with SNAP (100 μM). *, P < 0.05 (control/TIP39 (+) L-NA vs. TIP39/SNAP) [control: n = 15; TIP39: n = 37; TIP39 (+) L-NA: n = 6; SNAP: n = 8].
Under normal conditions only small amounts of cGMP in CMCs were detectable (0.325 ± 0.046 pg/μg). In the presence of TIP39 (10 nM), the cGMP level increased about 7-fold (2.377 ± 0.193 pg/μg). In copresence of L-NA we could not observe such an increase of the cGMP level with TIP39 (0.381 ± 0.045 pg/μg). As a positive control, the NO donor SNAP was used. SNAP (100 μmol/liter) was able to increase the cGMP level in the same range as TIP39 (2.498 ± 0.753 pg/μg).
Discussion
Expression of TIP39 in the cardiovascular system was confirmed. In contrast to PTHrP, TIP39 had no vasodilatory properties, but a strong negative inotropic effect was observed in the presence of L-NA. Under basal conditions TIP39 induces only a small negative inotropic effect. This observation led to the assumption that NO contributes to this effect and influences the contractility in a positive manner. The experiments on CMCs yielded similar results as experiments with isolated hearts but with a maximum effect at a lower concentration. These results suggest a direct effect of TIP39 on the cellular level and that myocyte-derived NO rather than vascular NO is responsible for this effect. Furthermore, we demonstrated that NO mediated its positive inotropic effect via cGMP.
Expression of the PTH2r in the cardiovascular system (3) has led to the assumption that TIP39, its natural ligand, might also be expressed in this system. Therefore, we examined the expression of TIP39 in different compartments of the cardiovascular system. We demonstrated TIP39 expression in the aorta and kidney. These findings confirm earlier observations (15). Furthermore, we detected TIP39 expression in the atria, left and right ventricles, isolated CMCs, and CECs. From our data, we were not able to determine the sources of TIP39 in the cardiovascular system precisely. Several sources seem to be possible.
The first possibility is endothelial cells. These cells express TIP39 mRNA in same range as other cells in the cardiovascular system. Therefore, we could not exclude that the endothelium is a potential source of TIP39. That is not unlikely because these cells are a source for many other mediators like NO, prostaglandin I2, or endothelin-1. Furthermore, it seems to be plausible that TIP39 acts also in a paracrine manner as the above-mentioned mediators. In this way TIP39 could exert vasodilating properties under pathophysiological conditions because our results have shown no effect under basal conditions. This assumption was supported by results from Usdin et al. (3), who demonstrated an expression of large amounts of PTH2r mRNA in the vasculature. TIP39 could reach CMCs via this possible paracrine release, too.
Second, CMCs themselves could release TIP39 in an autocrine or paracrine manner. There is strong evidence for such a release for PTHrP, another member of this family (26). PTHrP operates like TIP39 as a modulator of the contractile machinery of the heart (6). Indeed, all of the investigated tissues contained endothelial cells, so it is possible that the positive expression we observed in these tissues are from only the endothelium itself. However, our results from isolated CMCs, in which we also demonstrated an expression of TIP39, suggest that the myocardium itself is a source for TIP39.
Third, TIP39 was released from neuronal structures of the heart. Such a possibility would not be unlikely because TIP39 was initially described as a possible new neurotransmitter. It was first found in thalamic, midbrain, and pontine cell groups and some hypothalamic cells (27, 28) and seems to be involved in nociception (17). This hypothesis was supported by our finding that TIP39 is expressed in the ganglion stellatum. As mentioned above, we could not exclude that this expression was a result of endothelial cells contained in the preparation. Possible neuronal sources of TIP39 in the heart are terminals of postganglionic fibers or cardiac ganglion cells.
In conclusion, we were not able to identify the source of TIP39 in the heart. Furthermore, there is nothing known about the regulation of its expression or possible release mechanisms. Further studies have to investigate these open questions.
Expression and function of TIP39 outside the central nervous system was first shown in mammals by Eichinger et al. (15). Their data suggested a vasodilatory action of TIP39 in renal vessels at concentrations of 100 nM and 1 μM. These observations prompted us to investigate a possible role for TIP39 as a vasodilator in the cardiovascular system. However, TIP39 did not change coronary resistance. In contrast, we observed a small negative inotropic effect mediated by TIP39. Under basal conditions used in this preparation (saline perfusion), coronary resistance was low. Thus, a possible vasodilatory effect of TIP39 was seen more easily on preconstriction by L-NA. Again, we did not observe any vasodilatory effect but a strong negative inotropic effect. It seems that a blockade of the NO signaling pathway with L-NA unmasks this negative inotropic effect. If this is right, TIP39 affects the contractility of the heart in two different manners: one pathway depends on NO and exerts a positive contractile effect, and the second pathway mediates a strong negative inotropic effect. The cellular mechanisms contributing to this negative effect are still unknown.
The influence of NO on contractility of hearts has been controversial. Early investigations of NO effects indicated a negative inotropic effect (29, 30), but recent publications (31, 32, 33) suggested that NO has positive inotropic properties as well. Hirota et al. (34) showed that physiological concentrations of NO (0.16–1.7 μM) increase contractility in rat ventricular myocytes. The main target of NO in the cardiovascular system is guanylyl cyclase. This enzyme catalyzes the production of cGMP: high concentrations of cGMP activate the protein kinase G signaling pathway and exert a negative inotropic effect. Low concentrations lead to an induction of the cAMP/protein kinase A signaling pathway (34) and exert a positive inotropic effect. These findings prompted us to investigate whether TIP39 exerts a small, positive contractile effect via an activation of the NO/cGMP pathway. In fact, TIP39 increased cGMP levels in CMCs. The maximum effect of TIP39 on cGMP is comparable with that evoked by SNAP. At this concentration SNAP exerts a positive contractile effect. In the presence of L-NA, TIP39 did not increase cGMP levels. These findings support the hypothesis that TIP39 induces NO production and stimulates guanylyl cyclase via NO. Furthermore, our results on whole-heart preparations and single cells suggest that the source of NO that contributes to these effects is the CMCs themselves and not the endothelium.
All current publications describe two different important signaling pathways that are activated by TIP39: first, the cAMP/protein kinase A and second, the phospholipase C signaling pathway with elevation of intracellular [Ca2+] (13).
Both pathways have a common end point: an increase of cytosolic [Ca2+]. Therefore, we investigated in a preliminary experiment whether TIP39 alters intracellular Ca2+ concentrations. First results do not support this hypothesis. TIP39 in the presence of L-NA had no influence on the fura-2 ratio, which correlates with cytosolic Ca2+ levels (data not shown). Therefore, we found no indication for an activation of either the cAMP- or phospholipase C-dependent pathway in CMCs.
With this in mind, it seems not unlikely that a receptor that does not couple to these pathways is responsible for the negative inotropic effect. To identify the receptor subtype involved in the TIP39 response, we used the selective PTH2 receptor antagonist Trp23-Tyr36-PTHrP. A simultaneous blockade of the PTH1r by PTHrP(7–34) was necessary because Trp23-Tyr36-PTHrP is a potent activator of the PTH1r. The change of the amino acid at position 23 from Phe to Trp enables PTHrP to bind with high affinity to PTH2r (24). The competitive inhibition of the PTH1r was obvious because the increase in heart rate that is observed normally with Trp23-Tyr36-PTHrP was significantly reduced. Furthermore, we did not see an influence of PTHrP (natural ligand of PTH1r) on LVDP under basal conditions. In the presence of L-NA and the two receptor antagonists, we did not observe a decrease of LVDP by TIP39. These results suggest that this TIP39 effect is mediated via the PTH2r. Despite these obvious results, we could not exclude that both peptides activated another, yet-unknown receptor that has the same affinity for TIP39 as its antagonist Trp23-Tyr36-PTHrP.
In summary, our observations lead to the hypothesis that TIP39 activates two pathways. One signaling pathway is NO/cGMP dependent and influences inotropy in a positive manner. A second pathway can be seen on blockade of NO synthesis by L-NA. It seems very unlikely that the observed negative effect was also mediated via NO. In that case, we would not have seen any influence of TIP39 on contractility in the presence of L-NA. Therefore, we conclude that NO is responsible for the positive effect of TIP39 on contractility.
From this background, the following question arises: what conditions lead to a down-regulation of NO synthesis in the heart? Depre et al. (35) described an increase of neuronal NO synthase (NOS) within minutes under ischemic conditions. However, with prolonged ischemia, NOS expression decreased (36, 37). Also, increased tissue acidosis attenuated NOS activity (37). Nearly the same course of NO increase and decrease was observed during reperfusion (36). A similar biphasic regulation of NOS was observed in cardiac and vascular tissues under hypoxia (38). In conclusion, it seems not unlikely that TIP39 acts as a negative inotropic mediator. Future investigations should be directed toward verifying a possible involvement of TIP39 in this disease process. Furthermore, the signaling pathway mediating the strong negative inotropic effect of TIP39 needs to be established.
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Address all correspondence and requests for reprints to: Dr. Günter Ross, Institute of Physiology, University of Giessen, Aulweg 129, D-35392 Giessen, Germany. E-mail: guenter.ross@physiologie.med.uni-giessen.de.
Abstract
Tuberoinfundibular peptide (TIP39) was initially identified as a neurotransmitter and agonist of the PTH2 receptor, which is expressed in the cardiovascular system. This study documents for the first time the cardiac expression and function of TIP39.
Expression was analyzed via RT-PCR. Function was characterized on Langendorff-perfused rat hearts as left ventricular developed pressure (LVDP) and on isolated cells via a cell edge detection system. cGMP levels were detected with RIA. Tuberoinfundibular peptide (TIP39) mRNA was found to be constitutively expressed in coronary endothelium cells, isolated cardiomyocytes, ventricles, atria, and aorta. At first we investigated the vasodilatory properties of TIP39 (100 nM) in the presence of L-nitro-arginine (L-NA, 100 μM). Surprisingly, TIP39 had no vasodilatory effect but decreased LVDP by 35 ± 7%. In the absence of L-NA, addition of TIP39 decreased LVDP by 8 ± 2%. The PTH2 receptor antagonist Trp23-Tyr36-PTHrP (1–36, 100 nM) abolished this TIP39 effect in the presence of L-NA. The experiments with isolated cardiomyocytes provided similar results. TIP39 (10 nM) lowered the contraction amplitude by 6 ± 3%. In the presence of L-NA (100 μmol/liter), TIP39 lowered the amplitude by 34 ± 6%. cGMP concentration in cardiomyocytes was stimulated by TIP39 (10 nM) in the same range as by the nitric oxide (NO) donor SNAP (100 μM). In the presence of L-NA, this increase was abolished. These results suggest that an inhibition of endogenous NO production unmasks a profound negative inotropic effect of TIP39 that is mediated by an activation of the PTH2 receptor. The results obtained with isolated cardiomyocytes suggest that myocyte-derived NO, rather than vascular NO, is responsible for this effect. cGMP seems to be the downstream signal of produced NO.
Introduction
CURRENTLY DIFFERENT PTH receptors in mammals are known, namely the PTH1 receptor (PTH1r) and the PTH2 receptor (PTH2r). The expression of both receptors has been described in the cardiovascular system (1, 2, 3). These receptors are members of the same subgroup of the superfamily of heptahelical G protein-coupled receptors (4). The PTH2r has a homology of 51% to the PTH1r in its amino acid sequence (2).
The natural ligands of the PTH1r are PTHrP and PTH. Whereas PTH acts as a systemic mediator, which is synthesized from the parathyroid glands, PTHrP developed its properties in a paracrine or intracrine manner. PTH is known as a regulator of calcium homeostasis. PTHrP was first mentioned in combination with the clinical diagnosis of humoral hypercalcemia of malignancy. This syndrome is a common complication of lung and certain other cancers (5). Recently PTHrP was also identified as a factor with local cell regulatory properties. PTHrP can be released from these tissues under various physiological and pathophysiological conditions. In the cardiovascular system, for instance, PTHrP modulates heart rate, contraction, and vascular tonus (6). These effects have been linked to the PTH1r.
The PTH2r discriminates between PTH and PTHrP. Previous studies had described activation only by PTH (7, 8). However, Usdin et al. (9) isolated another peptide from the bovine hypothalamus that binds and activates this receptor. This peptide consists of 39 residues and has been designated tuberoinfundibular peptide (TIP39). A comparison among the sequences of TIP39, PTH, and PTHrP suggests that these peptides are all relatives (10).
Furthermore, phylogenetic analyses suggest that TIP39 is the archetype from which PTH and PTHrP was developed (11). Although PTH binds and activates the PTH2r, TIP39 seems to be the natural ligand of this receptor because PTH binds only with a low affinity to rat PTH2r, weakly stimulates accumulation of cAMP, and has no influence on intracellular calcium (12, 13). Another argument for this assumption is that the PTH2r is highly expressed in pancreas and brain, although the brain is a compartment with no detectable levels of PTH (3). Various tissues of human, e.g. brain, spinal cord, kidney, or heart (14), and rat, e.g. aorta, brain, renal vessels, kidney, and pancreas (15), express TIP39 mRNA, but its physiological role is presently unclear. In hypothalamus preparations an addition of TIP39 (100 nM) increased the release of many hypothalamic-releasing factors, e.g. CRH (16). Furthermore, TIP39 is released from supraspinal fibers that are involved in nociception. Peripherally injected TIP39 was able to stimulate nociceptors (17). It seems that TIP39 acts as not only a neuropeptide but also a vasoactive substance in renal vessels (15). The vasoactive properties are concentration dependent. A concentration of 1 nM results in vasodilatation, 10 nM leads to vasoconstriction, and 100 nM results in vasodilatation again.
Because the effects of the other members of the PTH family in cardiomyocytes are well documented, it is of great interest to know whether TIP39, the new member of this family, has any properties in this system and whether it is expressed in cardiac cells.
Furthermore, the question of whether an activation of the PTH2r by TIP39 on cardiac cells activates the same pathways described in the literature for other cells must be answered. The aim of this study was to investigate these open questions.
Materials and Methods
Experimental animals
Wistar rats weighting 200–250 g were used in the experiments. All animal studies were performed in accordance with guidelines described in the National Institutes of Health Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (publication no. 85–23, revised 1996). The animals were kept under standardized conditions of temperature, humidity, and light. They had free access to standard diet and drinking water ad libitum.
Cell culture
Cardiomyocytes (CMCs) and coronary endothelial cells (CECs) were obtained from rats (18). CECs were cultured as previously described (19, 20). Briefly, hearts were perfused with collagenase; the ventricles were chopped and dissolved in suspension. This suspension contained CMCs as well as CECs. To separate the cell types, the suspension was centrifuged. The supernatant contained the CECs, whereas the CMCs were in the sediment. To obtain CMCs, the sediment was resuspended, the cells were plated, and in a following step cultured at 37 C. After 2 h, cells were harvested. These cells were subsequently used for RNA extraction or cell contraction experiments. To obtain CECs, the supernatant was trypsinized and plated at a density of 106 cells on 100-mm plastic dishes. The cells were cultured at 37 C in medium 199 with Earle’s salt supplemented with penicillin G (100 IU/ml), streptomycin (100 μg/ml), and fetal bovine serum (10% vol/vol). The medium was renewed every second day.
After 5 d, when the cells had grown to confluence, they were trypsinized in PBS. Afterward the cells were prepared and used for RNA extraction. The purity of these cultures was more than 95% endothelial cells. The remaining cells were contaminations with smooth muscle cells, pericytes, and fibrocytes. The growth of other nonendothelial cells was inhibited by trypsinization and the very rapid attachment and growth of endothelial cells on the culture dishes.
PCR
Total RNA was extracted from CMCs and CECs as well as kidney, ganglion stellatum, aorta, left and right ventricle, and atria using the TRIzol method according to the manufacturer’s protocol. To avoid genomic contamination, RNA was treated with RNase-free DNase according to the manufacturer’s protocol. Treatment was performed for 1 h at 32 C in a final volume of 50 μl, containing 10 μl RNA, 5.0 mM MgCl2 (Invitrogen Life Technologies, Carlsbad, CA), 40 U/μl rRNAsin (Promega, Mannheim, Germany), and 1 U/μl RNase-free DNase (Novagen, Madison, WI). Thereafter DNA was precipitated by phenol-chloroform. Reverse transcriptase reactions were performed for 1 h at 37 C in a final volume of 5 μl using 2 μg RNA, 100 ng oligo (dT; Roche Molecular Biochemicals, Mannheim, Germany), 8 U/μl rRNAsin (Promega), and 60 U/μl Moloney murine leukemia virus reverse transcriptase (Invitrogen Life Technologies). Aliquots (1.5 μl) of synthesized cDNA were used for PCR in a final volume of 10 μl containing 0.3 mM of primer pairs (Invitrogen Life Technologies), 0.4 mM deoxynucleotide triphosphates, 1.5 mM MgCl2, and 5 U/μl Taq-DNA-polymerase (Invitrogen Life Technologies).
The quality of cDNA was tested with hypoxanthine-guanine-phosphoribosyl-transferase (HPRT) as a housekeeping gene. The following cycle conditions were used for HPRT: after denaturation for 3 min at 94 C, PCR cDNA was amplified by 25 cycles with denaturation at 94 C for 30 sec, annealing at 63 C for 45 sec, polymerization at 72 C for 30 sec and a final extension at 72 C for 3 min. To detect the expression of TIP39, we used two different primer pairs that were previously described by Eichinger et al. (15) [primer I-sequences: 5'-CTA GCT GAC GAC GCG GCC TTT CG-3' (sense); 5'-GTC CAG TAG CAA CAG CTT CTG C-3' (antisense)] and Della Penna et al. (13) [primer II-sequences: 5'-CTT GGG TAG CCC CCT GTC TCG G-3' (sense); 5' GGG CGC GTC CAG TAG CAA CAG C-3' (antisense)]. For primer I we used the following cycle conditions: after denaturation for 3 min at 94 C, PCR cDNA was amplified by 35 cycles with denaturation at 94 C for 30 sec, annealing at 62 C for 30 sec, polymerization at 72 C for 30 sec, and a final extension at 72 C for 3 min. For primer II we used the following cycle conditions: after denaturation for 3 min at 94 C, PCR cDNA was amplified by 38 cycles with denaturation at 94 C for 30 sec, annealing at 67 C for 30 sec, polymerization at 72 C for 30 sec, and a final extension at 72 C for 3 min. Amplified products were separated by electrophoresis on 2% agarose gel containing 0.04 μl ethidium bromide in Tris-acetate EDTA buffer. PCR products were identified by their expected size of 131 bp (HPRT), 102 bp (TIP39, primer I), and 157 bp (TIP39, primer II). Agarose gels were recorded with a video system.
Heart perfusion
In vitro experiments were performed in isolated, saline-perfused rat hearts as described previously (21). The hearts were mounted on a Langendorff perfusion system in a temperature-controlled chamber (37 C). The chamber was filled with humidified air during perfusion of hearts at 37 C and constant pressure (50 mm Hg) for an initial stabilizing period of 10 min. Diastolic pressure was adjusted to 12 mm Hg. After the period of stabilization, the perfusion was switched to constant flow. Hearts were perfused with oxygenated saline medium [composition of the perfusate (millimoles per liter): 124.0 NaCl, 2.7 KCl, 0.4 NaH2PO4, 1.0 MgCl2, 1.8 CaCl2, 24.0 NaHCO3, and 5.0 glucose, gassed with Carbogen (95% O2 + 5%CO2) (pH 7.40]. During administration of the different substances, hearts were perfused according to the protocols specified below in Results.
During the experiments, the following parameters were monitored: heart rate, systolic pressure, diastolic pressure, left ventricular developed pressure (LVDP, defined as left ventricular end-systolic pressure minus left ventricular end-diastolic pressure), and aortic pressure. In each group, six hearts were investigated.
Single-cell contraction
Ventricular cardiomyocytes were isolated from male rat hearts and paced at a constant frequency of 2 Hz as described before (18, 22). Each preparation of ventricular cardiomyocytes used in this study contained a mixed population of cells from two rat heats to reduce the influence of intraindividual differences. In each of these preparations, basal contractile responsiveness was determined from three cells. Cells selected for analysis did not beat spontaneously.
On average, less than 15% of all cells showed spontaneous beating within 60 sec. From the remaining cells, those cells that beat regularly for 60 sec if they were paced with 1 Hz were selected. Cell shortening was monitored using a cell-edge detection system from which the following parameters were calculated using the software MUCEL (Heidelberg, Germany): cell shortening is expressed as percentage of systolic cell length relative to diastolic cell length (L/L). The kinetics of contraction are expressed as rate constants for maximal contraction (Cmax) and relaxation velocity (Rmax). They were calculated by normalizing maximal contraction and relaxation velocities to peak contraction. In each case, cells were constantly paced and five contractions were recorded every 15 sec. The average of these recordings was used as one data point. The measurements on individual cells were repeated four times, and the average of these four measurements was used as the average contractile performance of individual cells. From each preparation, three cells were analyzed for each condition, and the same experiments were repeated with other preparations.
cGMP
CMCs were isolated from rats as mentioned above. The cultured CMCs were stimulated with different agents and then disintegrated with ethyl alcohol (100%). The lysate was centrifuged (12,000 rpm, 2 min). The supernatant was used for cGMP determination and the pellet for analysis of protein content. cGMP was determined by RIA using the cGMP [3H] assay system (code TRK500, Amersham Biosciences, Freiburg, Germany) according to the manufacturer’s protocol (23). The protein content was measured as previously described (20). The cGMP concentration was normalized to cellular protein content.
Agents used
The following agents were used: PTHrP 1–34 (100 nM; Bachem, Torrance, CA); TIP39 (0.1, 10, 50, 100, 200 nM; Bachem); N-nitro-L-arginine (L-NA) (100 μM; Sigma-Aldrich, Munich, Germany); Trp23-Tyr36-PTHrP 1–36 (PTH2r-antagonist) (24) (100 nM; Bachem, Torrance, CA); PTHrP (7–34) (100 nM; Bachem); and S-nitoso-N-acetylpenicillamine (SNAP) (100 μM; Calbiochem, Darmstadt, Germany).
Statistics
Quantitative results are expressed as means ± SEM. In experiments with more than two groups, ANOVA was used for comparison with a Student-Newmann-Keuls test for post hoc analysis. In cases in which two groups were compared, conventional t tests were performed. P < 0.05 was used as a significant difference between groups.
Results
Expression of TIP39
Expression of TIP39 was investigated in ganglion stellatum, left ventricle, right ventricle, atria, CECs, aorta, kidney, and CMCs (Fig. 1). To ensure amplification of TIP39, we used two different primer pairs. The primer pair one (pair 1) amplified a 102-bp fragment and the pair two (pair 2) a 157-bp fragment. The HPRT primers amplified a product of 131 bp. In all tissues or cells mentioned, we demonstrated an expression of TIP39 by both primers.
FIG. 1. TIP39 expression in cardiovascular tissues and cells. Typical examples of ethidium bromide gels are shown. RT-PCR analysis of HPRT (housekeeping gene) and TIP39 gene expression in ganglion stellatum (GS), left ventricle (LV), right ventricle (RV), atrium (AT), CECs, aorta (A), kidney (K), and CMCs. In every gel, a negative control (C) was made. This negative control contained all PCR substances without RNA sample. Similar results were obtained in three to four independent tissues or cell preparations.
A negative control was included on each gel (all PCR components without RNA sample). These controls were always negative. With both primers, we observed a stronger expression of TIP39 in the left ventricle and kidney. The expression in other tissues and cells were weaker.
Influence of TIP39 on contraction of isolated hearts
We wanted to first investigate the ability of TIP39 to vasodilate coronary vessels. Therefore, the coronary vessels were preconstricted with L-NA (100 μM) for 20 min. As control we used PTHrP, a PTH1r agonist. An addition of PTHrP decreased aortic pressure within a few minutes (Fig. 2). However, TIP39 (100 nM) showed no influence on perfusion pressure (Fig. 2). Surprisingly, under these conditions, TIP39 decreased LVDP about 35% (from 109.9 ± 9.9 to 71.8 ± 6.8 mm Hg; n = 6; P < 0.05; Fig. 3). No decrease of LVDP was observed with PTHrP (data not shown).
FIG. 2. Influence of PTHrP or TIP39 on the aortic pressure (AP). After a period of stabilization (10 min), coronary vessels and the aorta were preconstricted by L-NA (100 μM; 20 min). PTHrP (100 nM) showed a complete vasodilation within 10 min. TIP39 (100 nM) had no influence on aortic pressure. *, P < 0.05 (PTHrP: pretreatment vs. posttreatment, n = 6 hearts in each group). The relative vasodilative effect was expressed as percent decreases of AP before treatment. Perfusion pressure was initially set to 50 mm Hg.
FIG. 3. Influence of TIP39 (100 nM) on LVDP in presence of L-NA. After a period of stabilization (10 min), hearts were perfused with L-NA for 20 min followed by treatment with TIP39 (see Fig. 2). *, P < 0.05 (TIP39 vs. control, n = 6 hearts in each group).
Next we determined the ability TIP39 (100 nM) to lower LVDP under basal conditions without pretreatment with L-NA. In the absence of L-NA, TIP39 decreased LVDP of isolated, saline-perfused rat hearts about 8% (from 103.8 ± 8.0 to 91.4 ± 3.6 mm Hg; n = 6; P < 0.05). This decrease reached a maximum after 5 min (Fig. 4).
FIG. 4. Influence of TIP39 (100 nM) on LVDP in absence of L-NA. After a period of stabilization (10 min), an addition of TIP39 decreased LVDP. *, Difference between the control and TIP39 group after the beginning of treatment (P < 0.05, n = 6 hearts in each group).
In the following experiments, we investigated different concentrations of TIP39 with regard to LVDP. Therefore, we performed experiments with and without pretreatment of L-NA.
We used concentrations from 0.1 to 100 nM TIP39. Without L-NA (Fig. 5), a concentration of 100 nM TIP39 decreased LVDP 8.1 ± 2.2% (P < 0.05). At a concentration of 50 nM, TIP39 developed only a very weak effect (decrease of 4.0 ± 3.3%). Lower concentrations of TIP39 had no effects. The concentration-response curve was repeated in the presence of L-NA (Fig. 5). A concentration of 50 nM TIP39 caused a significant decrease of LVDP (16.3 ± 0.8%, P < 0.05). An addition of 100 nM showed a maximum decrease of LVDP (34.7 ± 6.8%, P < 0.05). All subsequent experiments were performed at a concentration of 100 nM.
FIG. 5. Concentration-response relationship for TIP39 in the presence and absence of L-NA (100 μM) in isolated perfused rat hearts. In the first group (, Tip39 (–) L-NA) treatment of TIP39 (5 min) preceded a period of stabilization (10 min). In the second group (, TIP39 (+) L-NA), the stabilization period (10 min) followed the L-NA treatment for 20 min and after this, hearts were treated with TIP39 at different concentrations (0.1,10, 50, and 100 nM) in combination with L-NA. *, #, P < 0.05 vs. initial situation before TIP39 treatment (n = 6 hearts for each concentration). The relative inotropic effects are expressed as percent decrease of LVDP before TIP39 treatment.
The strong decrease of LVDP caused by TIP39 in the presence of L-NA was attenuated by cotreatment with both receptor antagonists (PTH2r-antagonist: Trp23-Tyr36-PTHrP(1–36), PTH1r-antagonist PTHrP(7–34) [124.0 ± 4.7 (before treatment) to 124.5 ± 3.0 mm Hg (after treatment); n = 6; P > 0.05; Fig. 6]. The peptide Trp23-Tyr36-PTHrP binds with a high affinity to the PTH2r without activating this receptor (24). Because this peptide also activates the PTH1r, we used the PTH1r-antagonist PTHrP(7–34) (25). This peptide binds to this receptor, but the activating domain for the cAMP pathway is lacking. Both peptides were perfused in the presence of L-NA for 5 min before and during treatment with TIP39.
FIG. 6. Presence of Trp23-Tyr36-PTHrP(1–36; PTH2r-antagonist, 100 nM) abolished the influence of TIP39 (100 nM; 10 min) on LVDP in the presence of L-NA and the PTH1r antagonist PTHrP(7–34). Perfusions were performed with a period of stabilization (10 min), after a period of treatment with L-NA (20 min) in the presence of both receptor antagonists [RA; Trp23-Tyr36-PTHrP)(1–36) and PTHrP(7–34)] followed by a treatment of TIP39 in the presence of above-mentioned substances.
Influence of TIP39 on contraction amplitude of isolated CMCs
In a subsequent experimental step, we investigated whether the influence of TIP39 on the contraction is a direct effect on CMCs. Therefore, we investigated a concentration-response relationship on the single cell level. Experiments were performed with and without L-NA. The contraction amplitude of cell shortening (L/L) was normalized to diastolic cell length (percent). The concentrations tested were 0.1–10 nM TIP39 (Fig. 7A).
FIG. 7. A and B, Concentration-response relationship for TIP39 in the presence and absence of L-NA (100 μM) in single-cell preparations (CMC) on L/L normalized to L/L of controls. The CMC was stimulated at 2 Hz. In the first group [, Tip39 (–) L-NA], cells were treated only with TIP39 [0.1 nM (n = 90), 1 nM (n = 63), and 10 nM (n = 63)]. In the second group [, TIP39 (+) L-NA], the treatment of cells with TIP39 preceded an incubation period of 20 min with L-NA. The cells were treated with different concentrations of TIP39 [0.1 nM (n = 63), 1 nM (n = 42), and 10 nM (n = 30)]. *, #, P < 0.05 vs. untreated [0.1 (n = 87), 1 (n = 66), and 10 nM (n = 54)] cells or cells treated only with L-NA [0.1 (n = 66), 1 (n = 39), and 10 nM (n = 26)], respectively (A). Comparison of the influence of TIP39 (10 nM) (n = 63) alone and in the presence of L-NA (100 μM) (n = 30) on L/L, Cmax, and Rmax. *, P < 0.05 vs. untreated (n = 54) cells or cells treated only with L-NA (n = 26), respectively. The relative effects expressed as percent decrease of L/L, Cmax, and Rmax of treated (TIP39 ± L-NA) vs. untreated (± L-NA) cells (B).
Without L-NA, only a small decrease of L/L of 13.5 ± 3.5% (P < 0.05) was observed at 1 nM TIP39. An even higher concentration showed only a trend (decrease of 6.2 ± 2.7%; P = 0.3). In the presence of L-NA, a concentration of 1 nM TIP39 showed a decrease of 24.4 ± 4.7% (P < 0.05). At a concentration of 10 nM TIP39, a maximum decrease of the contraction amplitude was achieved (34.0 ± 5.9%; P < 0.05).
This negative contractile effect went along with reduced maximal relaxation (Rmax, decrease: 19.8 ± 8.9%) and contraction velocities (Cmax, decrease: 16.4 ± 5.3%, Fig. 7B). In all subsequent single-cell experiments, a concentration of 10 nM TIP39 was used.
Figure 8, A and B show representative single-cell contractions of individual rat ventricular cardiomyocytes paced at 2 Hz in the different groups. The figures describe the change in cell length normalized to diastolic cell length. Figure 8A shows a contraction in the control () and TIP39 groups . TIP39 reduced the contraction amplitude in comparison with the control. Figure 8B shows the same results between the L-NA control () and TIP39+L-NA () groups.
FIG. 8. A and B, Representative single-cell recordings of the time course of the length of individual rat ventricular CMCs paced at 2 Hz in the different groups. L/L is expressed as percentage of systolic cell length relative to diastolic cell length. , Control; , TIP39 (A); , control (L-NA); , TIP39 (+) L-NA (B).
Detection of cGMP
cGMP is a well-known downstream target in the nitric oxide (NO) signaling cascade. Because our results suggest an interaction of the PTH2r with the NO cascade, we wanted to know whether the concentration of cGMP is increased in cardiomyocytes in the presence of TIP39 (Fig. 9).
FIG. 9. cGMP concentrations in response to TIP39 were normalized to the protein content of each dish of different preparations. Cells were treated with TIP39 (10 nM) alone and in the presence of L-NA (100 μM) with a preincubation period of 20 min. As positive control, cells were treated with SNAP (100 μM). *, P < 0.05 (control/TIP39 (+) L-NA vs. TIP39/SNAP) [control: n = 15; TIP39: n = 37; TIP39 (+) L-NA: n = 6; SNAP: n = 8].
Under normal conditions only small amounts of cGMP in CMCs were detectable (0.325 ± 0.046 pg/μg). In the presence of TIP39 (10 nM), the cGMP level increased about 7-fold (2.377 ± 0.193 pg/μg). In copresence of L-NA we could not observe such an increase of the cGMP level with TIP39 (0.381 ± 0.045 pg/μg). As a positive control, the NO donor SNAP was used. SNAP (100 μmol/liter) was able to increase the cGMP level in the same range as TIP39 (2.498 ± 0.753 pg/μg).
Discussion
Expression of TIP39 in the cardiovascular system was confirmed. In contrast to PTHrP, TIP39 had no vasodilatory properties, but a strong negative inotropic effect was observed in the presence of L-NA. Under basal conditions TIP39 induces only a small negative inotropic effect. This observation led to the assumption that NO contributes to this effect and influences the contractility in a positive manner. The experiments on CMCs yielded similar results as experiments with isolated hearts but with a maximum effect at a lower concentration. These results suggest a direct effect of TIP39 on the cellular level and that myocyte-derived NO rather than vascular NO is responsible for this effect. Furthermore, we demonstrated that NO mediated its positive inotropic effect via cGMP.
Expression of the PTH2r in the cardiovascular system (3) has led to the assumption that TIP39, its natural ligand, might also be expressed in this system. Therefore, we examined the expression of TIP39 in different compartments of the cardiovascular system. We demonstrated TIP39 expression in the aorta and kidney. These findings confirm earlier observations (15). Furthermore, we detected TIP39 expression in the atria, left and right ventricles, isolated CMCs, and CECs. From our data, we were not able to determine the sources of TIP39 in the cardiovascular system precisely. Several sources seem to be possible.
The first possibility is endothelial cells. These cells express TIP39 mRNA in same range as other cells in the cardiovascular system. Therefore, we could not exclude that the endothelium is a potential source of TIP39. That is not unlikely because these cells are a source for many other mediators like NO, prostaglandin I2, or endothelin-1. Furthermore, it seems to be plausible that TIP39 acts also in a paracrine manner as the above-mentioned mediators. In this way TIP39 could exert vasodilating properties under pathophysiological conditions because our results have shown no effect under basal conditions. This assumption was supported by results from Usdin et al. (3), who demonstrated an expression of large amounts of PTH2r mRNA in the vasculature. TIP39 could reach CMCs via this possible paracrine release, too.
Second, CMCs themselves could release TIP39 in an autocrine or paracrine manner. There is strong evidence for such a release for PTHrP, another member of this family (26). PTHrP operates like TIP39 as a modulator of the contractile machinery of the heart (6). Indeed, all of the investigated tissues contained endothelial cells, so it is possible that the positive expression we observed in these tissues are from only the endothelium itself. However, our results from isolated CMCs, in which we also demonstrated an expression of TIP39, suggest that the myocardium itself is a source for TIP39.
Third, TIP39 was released from neuronal structures of the heart. Such a possibility would not be unlikely because TIP39 was initially described as a possible new neurotransmitter. It was first found in thalamic, midbrain, and pontine cell groups and some hypothalamic cells (27, 28) and seems to be involved in nociception (17). This hypothesis was supported by our finding that TIP39 is expressed in the ganglion stellatum. As mentioned above, we could not exclude that this expression was a result of endothelial cells contained in the preparation. Possible neuronal sources of TIP39 in the heart are terminals of postganglionic fibers or cardiac ganglion cells.
In conclusion, we were not able to identify the source of TIP39 in the heart. Furthermore, there is nothing known about the regulation of its expression or possible release mechanisms. Further studies have to investigate these open questions.
Expression and function of TIP39 outside the central nervous system was first shown in mammals by Eichinger et al. (15). Their data suggested a vasodilatory action of TIP39 in renal vessels at concentrations of 100 nM and 1 μM. These observations prompted us to investigate a possible role for TIP39 as a vasodilator in the cardiovascular system. However, TIP39 did not change coronary resistance. In contrast, we observed a small negative inotropic effect mediated by TIP39. Under basal conditions used in this preparation (saline perfusion), coronary resistance was low. Thus, a possible vasodilatory effect of TIP39 was seen more easily on preconstriction by L-NA. Again, we did not observe any vasodilatory effect but a strong negative inotropic effect. It seems that a blockade of the NO signaling pathway with L-NA unmasks this negative inotropic effect. If this is right, TIP39 affects the contractility of the heart in two different manners: one pathway depends on NO and exerts a positive contractile effect, and the second pathway mediates a strong negative inotropic effect. The cellular mechanisms contributing to this negative effect are still unknown.
The influence of NO on contractility of hearts has been controversial. Early investigations of NO effects indicated a negative inotropic effect (29, 30), but recent publications (31, 32, 33) suggested that NO has positive inotropic properties as well. Hirota et al. (34) showed that physiological concentrations of NO (0.16–1.7 μM) increase contractility in rat ventricular myocytes. The main target of NO in the cardiovascular system is guanylyl cyclase. This enzyme catalyzes the production of cGMP: high concentrations of cGMP activate the protein kinase G signaling pathway and exert a negative inotropic effect. Low concentrations lead to an induction of the cAMP/protein kinase A signaling pathway (34) and exert a positive inotropic effect. These findings prompted us to investigate whether TIP39 exerts a small, positive contractile effect via an activation of the NO/cGMP pathway. In fact, TIP39 increased cGMP levels in CMCs. The maximum effect of TIP39 on cGMP is comparable with that evoked by SNAP. At this concentration SNAP exerts a positive contractile effect. In the presence of L-NA, TIP39 did not increase cGMP levels. These findings support the hypothesis that TIP39 induces NO production and stimulates guanylyl cyclase via NO. Furthermore, our results on whole-heart preparations and single cells suggest that the source of NO that contributes to these effects is the CMCs themselves and not the endothelium.
All current publications describe two different important signaling pathways that are activated by TIP39: first, the cAMP/protein kinase A and second, the phospholipase C signaling pathway with elevation of intracellular [Ca2+] (13).
Both pathways have a common end point: an increase of cytosolic [Ca2+]. Therefore, we investigated in a preliminary experiment whether TIP39 alters intracellular Ca2+ concentrations. First results do not support this hypothesis. TIP39 in the presence of L-NA had no influence on the fura-2 ratio, which correlates with cytosolic Ca2+ levels (data not shown). Therefore, we found no indication for an activation of either the cAMP- or phospholipase C-dependent pathway in CMCs.
With this in mind, it seems not unlikely that a receptor that does not couple to these pathways is responsible for the negative inotropic effect. To identify the receptor subtype involved in the TIP39 response, we used the selective PTH2 receptor antagonist Trp23-Tyr36-PTHrP. A simultaneous blockade of the PTH1r by PTHrP(7–34) was necessary because Trp23-Tyr36-PTHrP is a potent activator of the PTH1r. The change of the amino acid at position 23 from Phe to Trp enables PTHrP to bind with high affinity to PTH2r (24). The competitive inhibition of the PTH1r was obvious because the increase in heart rate that is observed normally with Trp23-Tyr36-PTHrP was significantly reduced. Furthermore, we did not see an influence of PTHrP (natural ligand of PTH1r) on LVDP under basal conditions. In the presence of L-NA and the two receptor antagonists, we did not observe a decrease of LVDP by TIP39. These results suggest that this TIP39 effect is mediated via the PTH2r. Despite these obvious results, we could not exclude that both peptides activated another, yet-unknown receptor that has the same affinity for TIP39 as its antagonist Trp23-Tyr36-PTHrP.
In summary, our observations lead to the hypothesis that TIP39 activates two pathways. One signaling pathway is NO/cGMP dependent and influences inotropy in a positive manner. A second pathway can be seen on blockade of NO synthesis by L-NA. It seems very unlikely that the observed negative effect was also mediated via NO. In that case, we would not have seen any influence of TIP39 on contractility in the presence of L-NA. Therefore, we conclude that NO is responsible for the positive effect of TIP39 on contractility.
From this background, the following question arises: what conditions lead to a down-regulation of NO synthesis in the heart? Depre et al. (35) described an increase of neuronal NO synthase (NOS) within minutes under ischemic conditions. However, with prolonged ischemia, NOS expression decreased (36, 37). Also, increased tissue acidosis attenuated NOS activity (37). Nearly the same course of NO increase and decrease was observed during reperfusion (36). A similar biphasic regulation of NOS was observed in cardiac and vascular tissues under hypoxia (38). In conclusion, it seems not unlikely that TIP39 acts as a negative inotropic mediator. Future investigations should be directed toward verifying a possible involvement of TIP39 in this disease process. Furthermore, the signaling pathway mediating the strong negative inotropic effect of TIP39 needs to be established.
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