Parathyroid hormone-related protein-mediated responses in pulmonary arteries and veins of newborn lambs
Yuansheng Gao1,2 and
J. Usha Raj1
1Division of Neonatology, Los Angeles Biomedical Research Institute, Harbor-UCLA Medical Center, Geffen School of Medicine at University of California, Los Angeles, California; and 2Key Laboratory of Molecular Cardiovascular Sciences (Peking University), Ministry of Education, Beijing, China
Submitted 2 November 2004
; accepted in final form 28 February 2005
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ABSTRACT
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PTHrP has important roles in lung development and function. Here we determined the vasomotor responses of isolated pulmonary arteries and veins of newborn and adult sheep to PTHrP. In vessels constricted with endothelin-1, PTHrP (PTHrP 1-34) caused greater relaxation of veins than of arteries. In both vessel types, relaxation to the peptide was less in adult than in newborn vessels. In newborn lambs, PTHrP-induced relaxation was not affected by endothelium removal, inhibition of eNOS, or inhibition of adenylyl cyclases by SQ-22536. However, relaxation was attenuated by 4-aminopyridine, inhibitor of voltage-dependent potassium channels, in both arteries and veins, and by charybdotoxin, inhibitor of calcium-activated potassium channels, in veins. When vessels were saturated with 8-BrcAMP (3 x 104 M), to eliminate relaxation mediated by endogenous cAMP, PTHrP-induced relaxation was partially attenuated. In vessels treated with 8-BrcAMP (3 x 104 M), 4-aminopyridine but not charybdotoxin inhibited relaxation induced by PTHrP 1-34 in both arteries and veins. Radioimmunoassay showed that, in the presence of a general phosphodiesterase inhibitor, PTHrP caused a concentration-dependent increase in intracellular cAMP content in arteries and veins, which was largely abolished by SQ-22536. Our results demonstrate that PTHrP is a potent vasodilator of pulmonary vessels, with a greater effect in veins than in arteries. Relaxation induced by the peptide contains both cAMP-dependent and -independent components. In both arteries and veins, voltage-dependent potassium channels mediate the response to PTHrP, at least in part, in a cAMP-independent fashion; and in veins, calcium-activated potassium channels may be stimulated by elevated cAMP levels.
vasodilation; cAMP; potassium channel; perinatal lungs
PARATHYROID HORMONE-RELATED PROTEIN (PTHrP) was originally identified in tumors associated with the syndrome of humoral hypercalcemia of malignancy. Its term derives from the fact that the first 13 amino acids of PTHrP are highly homologous with those in the same position in parathyroid hormone (PTH) and both peptides act via PTH/PTHrP receptors. However, PTH is only produced by the parathyroid gland and the central nervous system, and its functions are primarily to maintain calcium homeostasis. In contrast, various cells and tissues produce PTHrP, and it has a broad range of functions (29, 37).
PTHrP is the product of a single gene. Through alternative splicing, it gives rise to three initial translation products: PTHrP 1-139, PTHrP 1-141, and PTHrP 1-173. These products undergo further posttranslational processing and give rise to a family of mature secretory forms of the peptide, each with its own physiological function and each with its own receptor or receptors (29, 37). There are three general functions of PTHrP, namely, regulation of transepithelial calcium transport; regulation of development, differentiation, and proliferation of tissues; and relaxation of smooth muscle cells (29, 37). The relaxant action of PTHrP resides in its 1-34 region (7, 29, 37) and has been demonstrated in a variety of smooth muscle cell types (18, 23, 24, 29, 33, 46, 47, 56). It is postulated that this peptide may induce relaxation of smooth muscle cells by increased production of intracellular cAMP (17, 18, 23, 24, 46). However, other mechanisms such as activation of potassium channels may also be involved. In porcine tracheal smooth muscle, PTHrP-induced relaxation is mediated in part by activation of large-conductance Ca2+-activated K+ (BKCa) channels (46). In mice, activation of ATP-sensitive K+ (KATP) channels in lymph vessels contributes to PTHrP-mediated inhibitory responses of lymphatic pump activity (28).
PTHrP appears to be essential for mammalian lung development since deletion of the PTHrP gene results in failed alveolarization (5, 53). It may also have an important role in regulating pulmonary function. Tonic stretching of type II alveolar epithelial cells stimulates the expression and production of PTHrP (54, 55). The peptide is a dilator of airway smooth muscle (46); however, its effect on pulmonary vasoactivity is not known. In this study the responses of ovine pulmonary arteries and veins to PTHrP and the mechanisms underlying the responses were studied.
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MATERIALS AND METHODS
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Twenty-six newborn lambs (711 days old, either sex) and six adult sheep (1 yr old, either sex) from Nebeker Ranch (Lancaster, CA) were used. They were anesthetized with ketamine hydrochloride (30 mg/kg im) and killed with an overdose of pentobarbital (iv). Animal handling and study protocols were reviewed and approved by the Los Angeles Biomedical Research Institute Animal Care and Use Review Committee.
The lungs were immediately removed, and fourth-generation pulmonary vessels were dissected free of parenchyma and cut into rings (length: 3 mm). The outside diameters of pulmonary arteries and veins of newborn lambs are 2.11 ± 0.14 and 1.69 ± 0.09 mm, respectively (n = 26 for each group); the outside diameters of the arteries and veins of adult sheep are 3.73 ± 0.31 and 3.61 ± 0.37 mm, respectively (n = 6 for each group). In some vessels, endothelium was mechanically removed by insertion of the tips of a watchmaker's forceps into the lumen of the vessel and rolling the vessel back and forth on saline-loaded filter paper. Removal of the endothelium was confirmed by lack of relaxation to acetylcholine (105 M) (15).
Organ chamber study.
Vessel rings were suspended in organ chambers filled with 10 ml of modified Krebs-Ringer bicarbonate solution [composition (in mM): 118.3 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25.0 NaHCO3, 11.1 glucose] and were maintained at 37 ± 0.5°C and aerated with 95% O2-5% CO2 (pH 7.4). Each ring was suspended via two stirrups that were passed through the lumen. One stirrup was anchored to the bottom of the organ chamber; the other one connected to a strain gauge (model FT03C; Grass Instrument, Braintree, MA) for the measurement of isometric force. The vessel tension was expressed as g/mm2 cross-sectional area of smooth muscle (CSASM) as previously described (13). It has been shown that the proportion of muscle in the total tissue cross-sectional area of the vessel is the most important factor for normalization of muscle tension (19).
At the beginning of the experiment, each vessel ring was stretched to its optimal resting tension. This was achieved by step-wise stretching, in 0.1-g increments, until the active contraction of the vessel ring to 100 mM KCl reached a plateau. The optimal resting tension was 0.63 ± 0.05 (n = 26) and 0.23 ± 0.08 g/mm2 CSASM for pulmonary arteries and veins of newborn lambs, respectively (n = 26 for each vessel type, P < 0.05). It was 0.33 ± 0.06 and 0.19 ± 0.04 g/mm2 CSASM for pulmonary arteries and veins of adult sheep, respectively (n = 6 each vessel type, P < 0.05).
After the tissues were brought to their optimal resting tension, 1 h of equilibration was allowed. Effect of PTHrP was determined in pulmonary vessels that were preconstricted with endothelin-1 to a similar tension. In some experiments, the effects of various pharmacological agents on PTHrP-induced responses were determined. We added these agents at least 45 min before testing their effects. In all experiments indomethacin (105 M) was present to prevent the possible interference of vasoactive prostanoids (14).
Radioimmunoassay of the intracellular content of cAMP.
Rings of ovine pulmonary arteries and veins were incubated in 10 ml of modified Krebs-Ringer bicarbonate solution (37°C, 95% O2 and 5% CO2) in the presence or absence of SQ-22536 [104 M, an inhibitor of adenylyl cyclases (16)]. To prevent the possible interference of prostanoids on cAMP formation, indomethacin (105 M) was present in all experiments (14). Forty-five minutes after the administration of solvent or the inhibitor, PTHrP 1-34 (1010 M107 M) was added into the vials. The vessels were freeze-clamped rapidly at different time points and thawed in trichloroacetic acid (6%), then homogenized in glass with a motor-driven Teflon pestle, sonicated for 5 s, and centrifuged at 13,000 g for 15 min. The supernatant was extracted with four volumes of water-saturated diethyl ether and lyophilized. The lyophilized samples were resuspended in 0.5 ml of sodium acetate buffer (0.05 M, pH 6.2), and their intracellular content of cAMP was determined using an assay kit purchased from Biomedical Technologies (Stoughton, MA). cAMP content is expressed as pmol/mg protein. The protein concentration of vessels was determined by the Bradford method with bovine serum albumin as standard (4).
Drugs.
The following drugs were used (unless otherwise specified, all were obtained from Sigma, St. Louis, MO): 4-aminopyridine, barium chloride, 8-bromo-adenosine 3'5'-cyclic monophosphate (8-BrcAMP), charybdotoxin (Biomol Research Laboratories, Plymouth Meeting, PA), endothelin-1 (American Peptide, Sunnyvale, CA), glibenclamide, indomethacin, isobutylmethylxanthine, L-norepinephrine hydrochloride, parathyroid hormone-related protein 1-34 (PTHrP 1-34; Peninsula Laboratories, Belmont, CA), SQ-22536, and DL-propranolol.
SQ-22536, glibenclamide, and isobutylmethylxanthine were dissolved in DMSO (final concentration: <0.2%). Preliminary experiments showed that DMSO at the concentrations used had no effect on contraction to endothelin-1 and had no effect on relaxation induced by PTHrP 1-34 in pulmonary vessels of newborn lambs. Indomethacin (105 M) was prepared in equal molar Na2CO3. This concentration of Na2CO3 did not significantly affect the pH of the solution in the organ chamber. Other drugs were prepared with distilled water.
Data analyses.
Data are shown as means ± SE. When mean values of two groups were compared, Student's t-test for unpaired observations was used. When the mean values of the same group before and after stimulation were compared, Student's t-test for paired observations was used. Comparison of mean values of more than two groups was performed with one-way ANOVA test with Student-Newman-Keuls test for post hoc testing of multiple comparisons. All these analyses were performed using a commercially available statistics package (Sigma Stat; Jandel Scientific, San Rafael, CA). Statistical significance was accepted when the P value (two tailed) was <0.05. In all experiments, n represents the number of animals.
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RESULTS
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Organ chamber studies.
We preconstricted pulmonary arteries and veins of the newborn lambs with endothelin-1 (3 x 109 M to 2 x 108 M) to comparable tension levels (1.46 ± 0.30 to 1.71 ± 0.26 g/mm2 CSASM) before testing the effect of PTHrP. These levels corresponded to 68.5 ± 2.9% and 46.9 ± 3.2% of the maximal contraction evoked by endothelin-1 for the arteries and veins, respectively (n = 36, P < 0.05).
After vessel tension was stable, PTHrP 1-34 was added in increasing concentrations. PTHrP induced marked relaxation of pulmonary veins with a threshold concentration of 3 x 1011 M. Arteries were less sensitive than veins to the peptide. Relaxation to PTHrP was not significantly different among vessels with endothelium, without endothelium, or with endothelium treated with nitro-L-arginine [104 M, an inhibitor of nitric oxide synthase (15)] (Fig. 1). Nitro-L-arginine was added before constriction of vessels with endothelin-1; it had no effect on basal tension in arteries, but it increased basal tension in veins by 0.83 ± 0.10 g/mm2 CSASM (n = 6, P < 0.05).

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Fig. 1. Relaxations of pulmonary arteries (PA) and veins (PV) of newborn lambs induced by parathyroid hormone-related protein (PTHrP) 1-34. Vessels were constricted to a similar tension with endothelin-1 (3 x 1096 x 109 M). Relaxation is expressed as percentage of vessel tension before the addition of PTHrP. E(+), with endothelium; E(), without endothelium; NLA, nitro-L-arginine, 104 M. Data are shown as means ± SE; n = 6 for each group. *Significant difference between arteries and veins (P < 0.05).
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The response of arteries and veins of the newborn lambs to PTHrP was also examined after the vessels were constricted with endothelin-1 using concentrations equivalent to EC50 and EC75 for endothelin-1, and with norepinephrine (105 M). Even under these conditions, when the absolute level of tension in arteries and veins was different, PTHrP induced a greater relaxation of veins than of arteries (Table 1). In pulmonary vessels of adult sheep constricted with endothelin-1 at EC50 concentrations [vessel tension: arteries, 8.36 ± 0.94 51g/mm2 CSASM; veins, 12.72 ± 1.51g/mm2 CSASM (n = 6, P < 0.05)], PTHrP caused a smaller relaxation in adult vessels than in vessels from newborn lambs; but similar to that in newborn lambs, in adult sheep, relaxation of pulmonary veins to PTHrP was significantly greater than that of arteries (Fig. 2).
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Table 1. Relaxation of pulmonary arteries and veins of newborn lambs induced by PTHrP 1-34 (107 M) after the vessels were constricted with endothelin-1 at EC50 and EC75 concentrations and with norepinephrine at 105 M
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Fig. 2. Relaxation of PA and PV of newborn lambs and adult sheep induced by PTHrP 1-34. Vessels were constricted with endothelin-1 at EC50 concentrations (2 x 1096 x 109 M). Relaxation is expressed as percentage of vessel tension before the addition of PTHrP. Data are shown as means ± SE; n = 6 for each group. *Significant difference between the newborn and adult vessels (P < 0.05).
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PTHrP may cause vasodilation by activation of adenylyl cyclase and elevation of intracellular cAMP levels (17, 18, 23, 24, 46). However, SQ-22536 (104 M), an inhibitor of adenylyl cyclases (16), had no significant effect on relaxation of pulmonary vessels induced by PTHrP 1-34 (Fig. 3). When the vessels were treated with 8-BrcAMP [3 x 104 M, a cell membrane-permeable analog of cAMP (25)], such that the tissues were saturated with 8-BrcAMP and that relaxation mediated by endogenous cAMP would be blocked, relaxation-induced by PTHrP 1-34 was moderately attenuated (Fig. 3). In vessels treated with 8-BrcAMP, a higher concentration of endothelin-1 (6 x 109 M to 2 x 108 M) was used to raise vessel tension to levels comparable to that in untreated vessels.

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Fig. 3. Relaxation of PA and PV induced by PTHrP 1-34 after treatment with SQ-22536 (104 M) or 8-bromo-adenosine 3'5'-cyclic monophosphate (8-BrcAMP, 3 x 104 M). Vessels were constricted to a similar tension with endothelin-1 (3 x 1092 x 108 M). Relaxation is expressed as percentage of vessel tension before the addition of PTHrP. Data are shown as means ± SE; n = 6 for each group. *Significant difference between the control and vessels treated with 8-BrcAMP (P < 0.05).
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The role of potassium channels in relaxation to PTHrP 1-34 was also studied. 4-Aminopyridine (3 x 103 M), a blocker of voltage-dependent potassium channels (31), significantly attenuated relaxation of both arteries and veins induced by the peptide. Charybdotoxin (107 M), a blocker of calcium-activated potassium channels (31), had no effect on the relaxation of arteries but caused a moderate inhibition in veins (Fig. 4). These potassium channel blockers were added before constriction of vessels with endothelin-1. 4-Aminopyridine increased the tension of arteries and veins by 0.46 ± 0.10 and 0.34 ± 0.11 g/mm2 CSASM, respectively (n = 8). Charybdotoxin had no effect on tension in arteries but increased that in veins by 0.15 ± 0.04 g/mm2 CSASM (n = 6). The effects of glibenclamide (105 M) and BaCl2 (3 x 105 M), blockers of KATP and inward rectifier potassium (KIR) channels (31), were also tested, and they did not significantly affect the responses to PTHrP (data not shown, n = 5 for each group).

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Fig. 4. Effects of 4-aminopyridine (4-AP, 3 x 103 M) or charybdotoxin (CTX, 107 M) on relaxation of PA and PV induced by PTHrP 1-34. Vessels were constricted to a similar tension with endothelin-1 (2 x 1096 x 109 M). Relaxation is expressed as percentage of vessel tension before the addition of PTHrP. Data are shown as means ± SE; n = 8 for each group. *Significant difference between the control and vessels treated with 4-AP for the top panel and significant difference between vessels treated with 4-AP and those treated with CTX for the bottom panel; significant difference between the control and vessels treated with charybdotoxin (P < 0.05).
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To determine whether or not cAMP is involved in the observed effects of 4-aminopyridine and charybdotoxin, these potassium blockers were tested in vessels pretreated with 8-BrcAMP (3 x 104 M). In vessels saturated with cAMP, 4-aminopyridine (3 x 103 M) significantly attenuated relaxation of both arteries and veins induced by PTHrP 1-34; however, charybdotoxin (107 M) had no effect on relaxation to this peptide (Fig. 5).

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Fig. 5. Effects of 4-AP (3 x 103 M) or CTX (107 M) on relaxation of PA and PV induced by PTHrP 1-34 after the vessels were pretreated with solvent or 8-BrcAMP (3 x 104 M). Vessels were constricted to a similar tension with endothelin-1 (2 x 109108 M). Relaxation is expressed as percentage of vessel tension before the addition of PTHrP. Data are shown as means ± SE; n = 8 for each group. *Significantly different from the control (vessels not treated with 8-BrcAMP); significantly different from vessels not treated with 4-AP or CTX (P < 0.05).
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cAMP.
The intracellular cAMP content in pulmonary arteries of newborn lambs was not significantly different from that in veins under basal conditions (Table 2). In the presence of isobutylmethylxanthine, a general inhibitor of phosphodiesterases (50), the cAMP content in both arteries and veins was significantly increased. There was a significant difference in cAMP levels between arteries and veins in the presence of isobutylmethylxanthine at 104 M but not at 103 M (Table 2).
PTHrP 1-34 (107 M) had no significant effect on cAMP levels in pulmonary vessels of newborn lambs under control conditions but caused a marked increase in the cyclic nucleotide level in the presence of isobutylmethylxanthine; the increase peaked at 3 min. There was a significant difference in the increase in cAMP levels in response to PTHrP between arteries and veins in the presence of isobutylmethylxanthine at 104 M (n = 6 for each group, P < 0.05) but not at 103 M (Fig. 6).

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Fig. 6. Effects of PTHrP 1-34 on the intracellular content of cAMP of PA and PV of newborn lambs at control conditions and in the presence of isobutylmethylxanthine (IBMX). Data are shown as means ± SE; n = 6 for each group. *Significantly different from control (P < 0.05); significantly different from vessels treated with IBMX at 104 M (P < 0.05).
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Increases in cAMP levels induced by PTHrP in pulmonary vessels were significantly attenuated by SQ-22536 (104 M), an inhibitor of adenylyl cyclase (16) (Fig. 7). SQ-22536 at higher concentrations (3 x 104 M and 103 M) did not cause any further inhibition of cAMP elevation caused by PTHrP (data not shown, n = 5 for each group, P > 0.05).

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Fig. 7. Effects of SQ-22536 (104 M) on the intracellular content of cAMP of PA and PV of newborn lambs at basal conditions and after stimulation with PTHrP 1-34. Experiments were conducted in the presence of IBMX (103 M). Data are shown as means ± SE; n = 6 for each group. *Significantly different from basal values (P < 0.05).
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Effect of PTHrP on the intracellular cAMP content in pulmonary vessels of adult sheep was compared with that in vessels of newborn lambs, in the presence of isobutylmethylxanthine at 103 M. PTHrP (107 M) caused a smaller elevation in cAMP levels in pulmonary vessels of adult sheep than in vessels of newborn lambs (level in adult vs. newborn arteries was 58.5 ± 6.1 vs. 117.2 ± 13.6 pmol/mg protein and in veins was 70.6 ± 7.2 vs. 95.1 ± 8.3; n = 6 for each group, P < 0.05).
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DISCUSSION
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PTHrP causes relaxation of smooth muscle in the airways (46), gastrointestinal tract (29), uterus (47), and bladder (56). PTHrP is a dilator of various blood vessels such as the aorta (18, 23) and renal (24) and coronary arteries (33). PTHrPs are endoproteolytically processed to yield a family of mature forms. The relaxant action of PTHrP resides in its 1-34 region (7, 29, 37). In the present study, PTHrP 1-34 relaxed both pulmonary arteries and veins of newborn and adult sheep but exhibited a greater potency in veins. Studies show that endothelin-1 has constrictor and dilator effects on the resistance pulmonary arteries of preterm lambs (22) and on pulmonary vessels of isolated, perfused lamb lungs (52). The dilator properties of endothelin-1 may be distinct for arteries and veins, thus contributing to the differential response between the arteries and veins. However, this seems unlikely. In midsized ovine pulmonary vessels as those used in the present study, we have previous shown that endothelin-1 does not have a dilator effect (51). The greater response of the veins to PTHrP vs. arteries was observed not only when the vessels were constricted to matched tension with endothelin-1 at different concentrations but also when they were constricted with endothelin-1 at EC50 and EC75 concentrations as well as with norepinephrine. Therefore, this phenomenon could not be an artifact of varied vessel tension, nor could it be a constrictor-specific event.
It is of importance to note that PTHrP induces relaxation of veins of newborn lambs at concentrations as low as 3 x 1011 M, which is within the physiological range of this peptide (42, 49). Furthermore, given the observation that this peptide induced greater relaxation of pulmonary vessels of the newborn lamb than of adult sheep, it is possible that PTHrP may play an important role in modulating pulmonary vasomotor tone and blood flow in the newborn period.
A variety of vasodilators may exert their effects by inducing release of endothelium-derived nitric oxide (EDNO) (10). In our study, relaxation induced by PTHrP was not affected by removal of the endothelium nor by nitro-L-arginine, an inhibitor of nitric oxide synthase (15). These results suggest that the effect of PTHrP on ovine pulmonary vessels is not endothelium dependent and that EDNO is not involved. In addition, although endothelin-1 was used to raise the vessel tension before the effect of PTHrP was tested, it is unlikely that relaxation induced by PTHrP was affected by release of nitric oxide from the endothelium by endothelin-1. Our previous study showed that endothelin-1 does not elicit EDNO-dependent responses in ovine pulmonary arteries and veins (51).
The involvement of EDNO in PTHrP effect varies with different species and different vascular beds. For instance, PTHrP causes the release of nitric oxide from endothelial cells of bovine pulmonary arteries (21). In mice, PTHrP-induced aortic relaxation is largely endothelium dependent, whereas an intact endothelium is not necessary for maximal portal vein relaxation (48). In rabbits, renal vasodilation induced by PTHrP is not endothelium dependent but is partially inhibited by methylene blue, a nonspecific inhibitor of soluble guanylyl cyclase (24).
Relaxation of smooth muscle caused by PTHrP is often accompanied by an elevation of cAMP, and hence, it is thought that the relaxant effect may be mediated by cAMP (17, 18, 23, 24, 46). In the present study, PTHrP had no significant effect on cAMP content of pulmonary vessels under control conditions but caused a marked elevation in cAMP levels in the presence of isobutylmethylxanthine, a general inhibitor of phosphodiesterases (50). The increase in cAMP level by PTHrP was similar in arteries and veins of newborn lambs when treated with isobutylmethylxanthine at 103 M. However, when treated with the phosphodiesterase inhibitor at a lower concentration (104 M), PTHrP caused a smaller increase in cAMP level in arteries than in veins. This suggests that arteries have greater phosphodiesterase activity than veins. Indeed, our previous study showed that this was the case in the perinatal pulmonary vasculature (34). Hence, the greater activity of phosphodiesterases may in part contribute to the smaller relaxation of arteries to PTHrP. Our study also shows that the peptide caused a smaller increase in cAMP content of pulmonary vessels of adult sheep than that of newborn lambs. This may in part contribute to the smaller response of pulmonary vessels of adult sheep to PTHrP.
The increase in intracellular cAMP levels induced by PTHrP in pulmonary vessels was significantly, although not completely, inhibited by SQ-22536, an inhibitor of adenylyl cyclase (16). The relaxation caused by PTHrP was not significantly affected. The underlying mechanism is not clear. To date, nine membrane-bound isoforms and one soluble form of adenylyl cyclase have been identified. Multiple isoforms may exist in the same cell and in distinct subcellular compartments such that cAMP generated in distinct microdomains may serve distinct cellular functions (36, 44). It has been reported that the effect of inhibitors of adenylyl cyclase such as SQ-22536 differs with different isoforms of the enzyme (20). Therefore, it is possible that the isoform of adenylyl cyclase that is involved in tension modulation may not be sensitive to SQ-22536 (1, 11, 57).
The role of cAMP in PTHrP-induced relaxation was also studied by treating the pulmonary vessels with 8-BrcAMP at very high concentrations (3 x 104 M). Under such conditions, relaxation induced by PTHrP would be blocked if it were mediated by cAMP. However, although the relaxation of both arteries and veins was moderately attenuated, it was not abolished, suggesting that cAMP-independent mechanisms may also contribute to the relaxation induced by PTHrP.
In blood vessels, four main classes of K+ channels have been described based on their biophysical and pharmacological properties, namely: KATP, KIR, BKCa, and voltage-gated K+ (KV) channels (26, 31, 43). In our study, relaxation of both pulmonary arteries and veins to PTHrP was inhibited by 4-aminopyridine, an inhibitor of KV channels (31). The inhibitory effect of 4-aminopyridine was also observed when the vessels were pretreated and saturated with 8-BrcAMP, a cAMP analog. This indicates that activation of KV channels by PTHrP may, at least in part, be cAMP independent. In pulmonary veins but not the arteries, PTHrP-induced relaxation was also inhibited by charybdotoxin, an inhibitor of BKCa channels (31). The effect of charybdotoxin was prevented by treatment with 8-BrcAMP at very high concentrations (3 x 104 M). Therefore, it appears as if PTHrP may exert its effect by activating BKCa channels via cAMP, which consequently results in membranous hyperpolarization, a reduction in calcium influx into the cell, and vasodilation. In pigs, relaxation of tracheal smooth muscle induced by PTHrP was attenuated by inhibitors of BKCa channels (46). In pulmonary vasculature of the rat and dog, cAMP-dependent activation of BKCa channels has also been reported (2, 3). Our results show that relaxation induced by PTHrP is not affected by glibenclamide and BaCl2, indicating that KATP and KIR channels are probably not involved (31).
In summary, our study demonstrates that in newborn lamb lungs, PTHrP is a potent dilator of pulmonary vessels, with the veins being more sensitive to the relaxant effect than arteries. Relaxation induced by PTHrP appears to contain a cAMP-dependent and a cAMP-independent component. In both arteries and veins, activation of Kv channels may play a role in the vasorelaxant effect of this peptide. Such an effect may be, at least in part, independent of the cAMP pathway. In veins but not in arteries, BKCa channels may contribute to the effect of PTHrP in a cAMP-dependent manner. Over the past two decades, a large amount of evidence has accumulated to indicate that pulmonary veins may play an important role in regulation of pulmonary circulation, particularly during the perinatal period and under certain pathophysiological conditions (12). Our data demonstrating that PTHrP is a potent dilator of newborn ovine pulmonary veins may implicate an important role for this peptide in modulating pulmonary vasoactivity in the perinatal period. In various cell types, including type II alveolar epithelial cells, mechanical stretch not only upregulates the expression of PTHrP but also acts as a potent stimulus for release of this peptide (6, 32, 39, 5456). Because pulmonary veins contribute significantly to total pulmonary vascular resistance in the fetus (8, 12, 40, 45), mechanical stretch of the lungs at birth may induce synthesis and release of PTHrP and promote veno-relaxation and a fall in pulmonary vascular resistance at birth. The expression and/or release of PTHrP are also augmented under a number of pathophysiological conditions such as ischemia, atherosclerosis, vascular stenosis, and various vasoconstrictors (9, 30, 32, 35, 38). Thus enhanced release of PTHrP in the lungs under certain pathophysiological conditions may act as a protective factor in reducing pulmonary vascular resistance (12, 40, 41).
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GRANTS
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This study was supported by National Heart, Lung, and Blood Institute Grants HL-059435 and HL-075187; by National Science Foundation of China Grants 30370523 and 30470629; and by Research Fund for Doctoral Programs of Higher Education (Ministry of Education, China) Grant 20030001031.
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ACKNOWLEDGMENTS
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We thank Dr. John S. Torday for valuable advice and Nik Phou for excellent secretarial assistance.
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FOOTNOTES
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Address for reprint requests and other correspondence: Y. Gao, Los Angeles Biomedical Research Institute, Harbor-UCLA Medical Center, 1124 W. Carson St., RB-1, Torrance, CA 90502 (E-mail: ysgao{at}labiomed.org)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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