Our objectives were to identify the relative contributions of [Ca2+]i and myofilament Ca2+ sensitivity in the pulmonary venous smooth muscle (PVSM) contractile response to the thromboxane A2 mimetic U-46619 and to assess the roles of PKC, tyrosine kinases (TK), and Rho-kinase (ROK) in that response. We tested the hypothesis that U-46619-induced contraction in PVSM is mediated by both increases in [Ca2+]i and myofilament Ca2+ sensitivity and that the PKC, TK, and ROK signaling pathways are involved. Isometric tension was measured in isolated endothelium-denuded (E−) canine pulmonary venous (PV) rings. In addition, [Ca2+]i and tension were simultaneously measured in fura-2-loaded E− PVSM strips. U-46619 (0.1 nM–1 μM) caused dose-dependent (P < 0.001) contraction in PV rings. U-46619 contraction was attenuated by inhibitors of L-type voltage-operated Ca2+ channels (nifedipine, P < 0.001), inositol 1,4,5-trisphosphate-mediated Ca2+ release (2-aminoethoxydiphenylborate, P < 0.001), PKC (bisindolylmaleimide I, P < 0.001), TK (tyrphostin A-47, P = 0.014), and ROK (Y-27632, P = 0.008). In PV strips, U-46619 contraction was associated with increases in [Ca2+]i and myofilament Ca2+ sensitivity. Both Ca2+ influx and release mediated the early transient increase in [Ca2+]i, whereas the late sustained increase in [Ca2+]i only involved Ca2+ influx. Inhibition of both PKC and ROK (P = 0.006 and P = 0.002, respectively), but not TK, attenuated the U-46619-induced increase in myofilament Ca2+ sensitivity. These results suggest that U-46619 contraction is mediated by Ca2+ influx, Ca2+ release, and increased myofilament Ca2+ sensitivity. The PKC, TK, and ROK signaling pathways are involved in U-46619 contraction.
- pulmonary vein
- Ca2+ homeostasis
- myofilament Ca2+ sensitivity
thromboxane a2 (TXA2) is a major product of arachidonic acid metabolism via the cyclooxygenase pathway. TXA2 is a potent stimulator of platelet aggregation and smooth muscle contraction and may play a role as a mediator of myocardial infarction, atherosclerosis, and bronchial asthma (22). Increased activity of TXA2 has also been implicated in several forms of human and experimental pulmonary hypertension, including pulmonary hypertension induced by sepsis (36), heparin/protamine (19), and ischemia-reperfusion (39).
The TXA2 analog, U-46619, is known to cause constriction of pulmonary veins (13). Pulmonary venous constriction can increase pulmonary capillary pressure and transvascular fluid flux to cause pulmonary edema, which can adversely affect blood oxygenation and right ventricular function. Agonist-induced vascular smooth muscle contraction is mediated by an increase in intracellular Ca2+ concentration ([Ca2+]i) and/or an increase in myofilament Ca2+ sensitivity. We are aware of only one study that has investigated the effects of TXA2 on [Ca2+]i in pulmonary veins (13). In that study, changes in tension in response to the TXA2 analog, U-46619, were measured in pulmonary venous rings, whereas changes in [Ca2+]i were measured separately in dispersed pulmonary venous smooth muscle cells (13). Although U-46619 was found to cause marked contraction in pulmonary venous rings, it failed to evoke any change in [Ca2+]i in a majority (56%) of the dispersed pulmonary venous smooth muscle cells (13). To our knowledge, simultaneous changes in tension and [Ca2+]i in response to TXA2 in pulmonary veins have never been reported. In the present study, we investigated the role of Ca2+ influx, inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ release, and myofilament Ca2+ sensitivity in response to the TXA2 analog, U-46619, in canine pulmonary veins while concomitantly measuring [Ca2+]i and tension. We also investigated the roles of the protein kinase C (PKC), Rho-kinase (ROK), and tyrosine kinases (TK) signaling pathways in U-46619 contraction of pulmonary veins.
MATERIALS AND METHODS
All experimental procedures and protocols were approved by the Institutional Animal Care and Use Committee of the Cleveland Clinic Foundation.
Preparation of pulmonary venous rings.
Healthy male mongrel dogs weighing 24–32 kg were anesthetized with intravenous pentobarbital sodium (30 mg/kg) and fentanyl citrate (15 μg/kg). After tracheal intubation, the lungs were mechanically ventilated. A catheter was placed in the right femoral artery, and the dogs were exsanguinated by controlled hemorrhage. A left lateral thoracotomy was performed through the fifth intercostal space, and the heart was arrested with induced ventricular fibrillation. The heart and lungs were removed from the thorax en bloc, and the lower right and left lung lobes were dissected free. Intralobar pulmonary veins [1–2 mm inner diameter (ID)] were carefully dissected and immersed in cold modified Krebs-Ringer bicarbonate (KRB) solution composed of 118.3 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 2.5 mM NaHCO3, 0.016 mM Ca-EDTA, and 11.1 mM glucose. The pulmonary veins were cleaned of connective tissue and cut into ring segments 4–5 mm in length. The endothelium was removed in all rings by gently rubbing the intimal surface with a cotton swab. Endothelial denudation abolished the vasorelaxant response to the endothelium-dependent vasodilator bradykinin (0.01 μM).
Isometric tension experiments.
Pulmonary venous rings were vertically mounted between two stainless steel hooks in organ baths filled with 25 ml of KRB solution (37°C) gassed with 95% O2 and 5% CO2. One of the hooks was anchored and the other was connected to a strain gauge to measure isometric force. The rings were stretched at 5-min intervals in increments of 0.5 g to achieve optimal resting tension. Optimal resting tension was defined as the minimal amount of stretch required to achieve the largest contractile response to 60 mM KCl and was determined in preliminary experiments to be 1.5 g. After the pulmonary venous rings had been stretched to their optimal resting tension, the contractile response to 60 mM KCl was assessed. After washout of KCl from the organ chamber and the return of isometric tension to prestimulation values, a concentration-response curve to U-46619 was performed in each ring. This was achieved by increasing the concentration of U-46619 in half-log increments (from 0.1 nM to 1 μM) after the response to each preceding concentration had reached a steady state.
To characterize the relative contribution of Ca2+ influx and Ca2+ release to the total contractile response to U-46619, pulmonary venous rings were pretreated with nifedipine (10 μM), an L-type voltage-operated Ca2+ channel inhibitor, and 2-aminoethoxydiphenylborate (2-APB, 100 μM), an IP3-mediated Ca2+ release inhibitor. After incubation with these inhibitors for 30 min, the rings were contracted with U-46619 (0.1 nM to 1 μM). The contractile responses to U-46619 were compared in untreated rings and paired rings treated with nifedipine or 2-APB.
To identify roles for the PKC, ROK, and TK signaling pathways in the contractile response to U-46619, pulmonary venous rings were incubated with either bisindolylmaleimide I (BIS1, 3 μM), a PKC inhibitor; Y-27632 (10 μM), an ROK inhibitor; or tyrphostin A-47 (10 μM), a TK inhibitor. The effect of combined inhibition of PKC, ROK, and TK was also assessed. The concentrations of inhibitors in the present study are within the range of concentrations used in previous studies (3, 4, 12). Pulmonary venous rings were pretreated with these inhibitors for 30 min before administration of U-46619. None of the inhibitors had an effect on baseline tension. In these isometric tension experiments, all the concentration-response curves were performed in paired size- and position-matched rings in the presence or absence of an inhibitor(s).
Preparation of pulmonary venous smooth muscle strips.
First-generation intralobular pulmonary veins (2∼4 mm ID) were carefully dissected and immersed in cold modified KRB solution. Compared with the ring studies, larger veins were used to simultaneously measure tension and [Ca2+]i to be compatible with the equipment used to make the fluorescence measurements (described below). The pulmonary veins were cleaned of connective tissue and cut into strips (2 × 8 mm). The endothelium was removed by gently rubbing the intimal surface with a cotton swab. Endothelial denudation was later verified by the absence of a vasorelaxant response to bradykinin (0.01 μM).
Simultaneous measurement of tension and [Ca2+]i.
Pulmonary venous strips without endothelium were loaded with 5 μM acetoxylmethyl ester of fura-2 (fura-2 AM) for 4 h at 37°C. A noncytotoxic detergent, 0.05% cremophor EL, was added to solubilize the fura-2 AM in the solution. After fura-2 loading, pulmonary venous strips were washed with KRB buffer to remove uncleaved fura-2 AM and mounted between two stainless steel hooks in a temperature-controlled (37°C) 3-ml cuvette. The strips were continuously perfused at 12 ml/min with the KRB solution bubbled with 95% O2 and 5% CO2 (pH 7.4). One hook was anchored, and the other was connected to a strain gauge transducer to measure isometric tension. The resting tension was adjusted to 0.5 g, which was determined in preliminary studies to be optimal for achieving a maximum contractile response to 40 mM KCl. We used 40 mM KCl (rather than 60 mM KCl) in the strip studies because the higher concentration was associated with a prolonged washout period before tension and [Ca2+]i returned to baseline values. Fluorescence measurements were performed with a dual-wavelength spectrofluorometer (Deltascan RFK6002; Photon Technology International, Lawrenceville, NJ) at excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm. Because calculations of absolute concentrations of [Ca2+]i rely on a number of assumptions, the 340/380 fluorescence ratio (340/380 ratio) was used as a measure of [Ca2+]i. The individual 340 and 380 signals were measured in all experiments, and the signals were observed to change in opposite directions in response to the various interventions. Because each pulmonary venous strip served as its own control, background fluorescence was assumed to be constant and was not subtracted from the 340/380 ratio. The temperature of all solutions was maintained at 37°C in a water bath. Fura-2 fluorescence signals (340 and 380 nm and 340/380 ratio) and tension were measured at a sampling frequency of 2 Hz and were collected with a software package from Photon Technology International.
Pulmonary venous strip experimental protocols.
We measured tension and [Ca2+]i simultaneously to investigate mechanisms of U-46619 contraction. In all protocols, responses were measured in paired size- and position-matched strips in the presence or absence of an inhibitor or treatment. Summarized data for changes in [Ca2+]i and tension are expressed as the percent change of the response to 40 mM KCl.
In protocol 1, pulmonary venous strips were first treated with 40 mM KCl. After increases in [Ca2+]i and tension reached new steady-state values (∼10 min), the strips were washed with fresh KRB solution, and [Ca2+]i and tension returned to baseline. U-46619 contraction was then assessed in the presence or absence of nifedipine (10 μM).
In protocol 2, pulmonary venous strips were treated with 40 mM KCl. After washout and return of [Ca2+]i and tension to baseline, U-46619 contraction was assessed in the presence (KRB solution) or absence of extracellular Ca2+ (2 mM EGTA), as well as in the presence or absence of 2-APB.
In protocol 3, we investigated the effects of U-46619 on the [Ca2+]i-tension relationship by incrementally increasing the extracellular Ca2+ concentration. In each pulmonary venous strip, the response to 40 mM KCl was assessed first. After washout, the strips were treated with a Ca2+-free buffer containing 2 mM EGTA for 10 min. This solution was replaced with a Ca2+-free buffer that did not contain EGTA. After 10 min, this solution was replaced with a Ca2+-free solution containing 40 mM KCl. Finally, after 10 min, the extracellular Ca2+ concentration was increased in control and U-46619-pretreated (100 nM) strips in an incremental fashion from 0 to 0.125, 0.25, 0.5, 1.25, and 2.5 mM. The same procedure was repeated in strips pretreated with BIS1 (3 μM), Y-27632 (10 μM), or tyrphostin A-47 (10 μM).
Solutions and chemicals.
All drugs were of the highest purity commercially available: U-46619 (Cayman Chemical, Ann Arbor, MI), nifedipine, 2-APB, Y-27632, tyrphostin A-47 (Calbiochem-Novabiochem, San Diego, CA), BIS1, cremophor EL, dimethyl sulfoxide (Sigma, St. Louis, MO), and fura-2 AM (Texas Fluorescence Labs, Austin, TX). U-46619, BIS1, nifedipine, and fura-2 AM were dissolved in dimethyl sulfoxide and diluted with distilled water. The final concentration of dimethyl sulfoxide in the organ bath and cuvette was <0.1% (vol/vol). None of the agents or solutions caused significant shifts in isometric tension or the 340/380 ratio at the concentrations used in these studies.
All data are expressed as means ± SD. The U-46619 contractile responses were compared in matched control and “treated” rings or strips from the same dog. Two-way analysis of variance for repeated measures followed by contrast analysis and Bonferroni correction were used for comparisons within and between groups. For ring studies, the U-46619 dose was used as the within-subject factor, and treatment (with or without) was used as the between-subject factor. For strips studies, extracellular Ca2+ concentration was used as the within-subject factor, and treatment (with or without) was used as the between-subject factor. Statistical analyses utilized SPSS for WINDOWS software (version 11.5; SPSS, Chicago, IL). Student's t-test for paired comparisons was used when two groups were compared. A P value of <0.05 was chosen as significant. In all experiments, sample size (n values) equals the number of dogs from which pulmonary venous rings or strips were taken.
Roles of Ca2+ influx and Ca2+ release in U-46619 contraction.
We tested the hypothesis that inhibition of Ca2+ influx and/or Ca2+ release would attenuate U-46619 contraction in pulmonary venous rings. Neither nifedipine nor 2-APB had an effect on resting tension. However, U-46619 contraction was attenuated (P < 0.001) by both nifedipine (Fig. 1A) and 2-APB (Fig. 1B).
Effects of U-46619 on simultaneous measurements of [Ca2+]i and tension.
To investigate the roles of Ca2+ influx and release on U-46619 contraction in more detail, we measured changes in [Ca2+]i and tension in response to 0.1 μM U-46619 in fura-2-loaded pulmonary venous strips. As illustrated in the original recordings in Fig. 2A, 40 mM KCl caused monotonic increases in [Ca2+]i and tension, whereas U-46619 caused early peak increases in [Ca2+]i and tension, followed by decreases in [Ca2+]i and tension to values below the peak but remaining well above baseline values. Early (corresponding to peak increases during the 5-min period following U-46619 administration) and late (measured 15 min after administrating U-46619) increases in [Ca2+]i and tension in response to 0.1 μM U-46619 are summarized in Fig. 2B. U-46619 caused approximately fourfold greater increases in tension compared with 40 mM KCl, whereas concomitant increases in [Ca2+]i in response to U-46619 were fairly similar to those induced by KCl. Pretreatment with nifedipine decreased the U-46619-induced early peak increases in tension and [Ca2+]i by 66 ± 4% and 20 ± 2%, respectively, and the late increases in tension and [Ca2+]i by 62 ± 5% and 67 ± 4%, respectively (Fig. 3A). In the absence of extracellular Ca2+, the early peak increases in tension and [Ca2+]i in response to U-46619 were decreased by 76 ± 5% and 31 ± 5%, the late increase in tension was decreased by 85 ± 9%, and the late increase in [Ca2+]i was abolished (Fig. 3B). After treatment with 2-APB in the absence of extracellular Ca2+, the early peak increase in [Ca2+]i in response to U-46619 was abolished, whereas early and late increases in tension were still apparent, although decreased by 50 ± 4% and 40 ± 5%, respectively, compared with the Ca2+-free condition (Fig. 3C).
Roles of PKC, ROK, and TK signaling pathways in U-46619 contraction.
We tested the hypothesis that inhibition of the PKC, ROK, and/or TK signaling pathways would attenuate U-46619 contraction. As summarized in Fig. 4, PKC inhibition with BIS1, ROK inhibition with Y-27632, and TK inhibition with tyrphostin A-47 all attenuated U-46619 contraction in pulmonary venous rings (P < 0.001, P = 0.008, and P = 0.014, respectively), although the inhibitory effect of tyrphostin A-47 was not apparent at higher concentrations of U-46619. Combined inhibition of the PKC, ROK, and TK pathways almost completely abolished U-46619 contraction (P < 0.001) (Fig. 5).
Effect of U-46619 on [Ca2+]i-tension relationship.
The observation in Fig. 3C that U-46619 still caused increases in tension without concomitant increases in [Ca2+]i suggests that U-46619 increases myofilament Ca2+ sensitivity. To test this hypothesis, control and U-46619-pretreated pulmonary venous strips bathed in a Ca2+-free buffer containing 40 mM KCl were exposed to incremental increases in extracellular Ca2+ concentration. As summarized in Fig. 6, U-46619 caused a leftward shift in the [Ca2+]i-tension relationship, such that for a given value of [Ca2+]i, tension was greater (P = 0.023) in the U-46619-pretreated strips compared with control (i.e., U-46619 increased myofilament Ca2+ sensitivity).
Roles of PKC, ROK, and TK signaling pathways in U-46619-induced increases in myofilament Ca2+ sensitivity.
We tested the hypothesis that inhibition of PKC, ROK, and/or TK would cause a rightward shift in the U-46619 [Ca2+]i-tension relationship. Using the same experimental design described above, we measured [Ca2+]i-tension relationships in U-46619-pretreated pulmonary venous strips in the absence and in the presence of signaling pathway inhibitors. As summarized in Fig. 7, BIS1 and Y-27632 each caused a rightward shift in the U-46619 [Ca2+]i-tension relationship (P = 0.006 and P = 0.002, respectively), whereas tyrphostin A-47 caused a leftward shift in the relationship (P = 0.037).
This is the first study to simultaneously measure changes in [Ca2+]i and tension in response to the TXA2 analog, U-46619, in pulmonary veins. Our results indicate that U-46619 contraction is mediated by: 1) Ca2+ influx via L-type voltage-gated Ca2+ channels; 2) IP3 receptor-mediated Ca2+ release from the sarcoplasmic reticulum; 3) an increase in myofilament Ca2+ sensitivity; and 4) activation of the PKC, ROK, and TK signaling pathways.
Mechanisms of U-46619 contraction have been investigated in a number of smooth muscle preparations. Although U-46619 is well known to cause contraction in pulmonary veins, most investigators have simply utilized U-46619 to precontract pulmonary veins before assessing responses to putative vasorelaxants (7, 8, 34). U-46619 has been shown to constrict human pulmonary veins harvested during lung cancer surgery via activation of TXA2-selective prostanoid receptors (35). Only one previous study has investigated intracellular mechanisms that could potentially mediate U-46619 contraction in pulmonary veins (13). The goal of the present study was to investigate the roles of both [Ca2+]i and myofilament Ca2+ sensitivity in the contractile response to U-46619 in pulmonary veins. It is important to note that results from studies of systemic vascular beds cannot easily be extrapolated to the pulmonary circulation (e.g., response to hypoxia). Moreover, results from pulmonary arteries cannot necessarily be extrapolated to pulmonary veins. In some species, U-46619 does not cause contraction of pulmonary arteries, whereas it causes marked contraction of pulmonary veins (13, 15).
Ca2+ influx and Ca2+ release.
We first examined the net effect of L-type Ca2+ channel inhibition (nifedipine) and IP3 receptor inhibition (2-APB) on the dose-response relationship to U-46619 in pulmonary venous rings. Both nifedipine and 2-APB significantly attenuated U-46619-induced increases in tension, causing a rightward and downward shift in the dose-response relationship. To investigate this in more detail and to confirm that the attenuating effect of these inhibitors was due to inhibition of Ca2+ influx and inhibition of Ca2+ release from the sarcoplasmic reticulum, we assessed the contractile response to U-46619 while simultaneously measuring [Ca2+]i in pulmonary venous strips. In control pulmonary venous strips, U-46619 contraction was associated with a reproducible and marked increase in [Ca2+]i. Nifedipine attenuated the contractile response to U-46619, and this was associated with a small decrease in the early peak increase in [Ca2+]i in response to U-46619 and a larger decrease in the late increase in [Ca2+]i in response to U-46619. U-46619 was also attenuated in strips treated with a Ca2+-free solution. This was associated with a concomitant decrease in the early peak increase in [Ca2+]i, whereas the late increase in [Ca2+]i was abolished. When pulmonary venous strips were treated with 2-APB in a Ca2+-free solution, U-46619 contraction was attenuated compared with that measured in the Ca2+-free solution alone, and this contractile response occurred without a concomitant change in [Ca2+]i. Our interpretation of all these results together is that: 1) U-46619 increases [Ca2+]i; 2) the early increase in [Ca2+]i primarily reflects IP3-mediated release of Ca2+ from the sarcoplasmic reticulum (although there may be a small Ca2+ influx component, as suggested by the nifedipine data); 3) the late increase in [Ca2+]i is entirely dependent on Ca2+ influx, and a component of this involves influx via L-type Ca2+ channels (note: receptor-operated Ca2+ channels and capacitative Ca2+ entry were not investigated); and 4) U-46619 contraction can be observed in the absence of an increase in [Ca2+]i. This last result suggests that U-46619 increases myofilament Ca2+ sensitivity.
Myofilament Ca2+ sensitivity.
U-46619-induced increases in [Ca2+]i were relatively similar to increases in response to 40 mM KCl, whereas the concomitant increase in tension in response to U-46619 was fourfold greater than KCl. After removal of extracellular Ca2+ and inhibition of IP3 receptor-mediated Ca2+ release, U-46619 increased tension without a concomitant increase in [Ca2+]i. Both of these results suggest that U-46619 increases myofilament Ca2+ sensitivity in pulmonary veins. To more directly investigate this possibility, we assessed the effects of U-46619 on the [Ca2+]i-tension relationship. U-46619 caused a marked leftward shift in the [Ca2+]i-tension relationship compared with control. Thus, for a given value of [Ca2+]i, tension was greater compared with control; i.e., U-46619 increased myofilament Ca2+ sensitivity in pulmonary veins.
Inhibition of PKC, ROK, and TK each attenuated the contractile response to U-46619 in pulmonary venous rings. Combined inhibition of these pathways almost completely abolished U-46619 contraction. Although we did not directly assess the roles of these signaling pathways on Ca2+ influx or Ca2+ release, we did test the hypothesis that the attenuating effect of these signaling pathway inhibitors was due to a reduction in the U-46619-induced increase in myofilament Ca2+ sensitivity. The involvement of PKC (1, 3, 4, 6, 9, 13, 14, 25, 30, 37), ROK (3–5, 10, 13, 25, 26), and TK (3, 4, 13, 23, 28) in Ca2+ sensitization has been reported in intact and permeabilized arteries from different species. PKC may modulate Ca2+ sensitivity via downregulation of myosin light chain phosphatase (MLCP) (18) or by phosphorylation of caldesmon and calponin (11, 16). PKC has been reported to play a role in U-46619-mediated vasoconstriction in rat pulmonary artery (2), rat aorta (31), and rabbit pulmonary artery (21). We observed that PKC inhibition caused a rightward shift in the [Ca2+]i-tension relationship, which supports the concept that the U-46619-induced increase in myofilament Ca2+ sensitivity involves the PKC signaling pathway. Consistent with our results, PKC inhibition has been reported to decrease U-46619 contraction via an effect on myofilament Ca2+ sensitivity in human omental arteries (17). Because PKC inhibition did not abolish U-46619 contraction (Fig. 4), we investigated other signaling pathways that could mediate a U-46619-induced increase in myofilament Ca2+ sensitivity.
Recent evidence suggests that activation of ROK by the active GTPase RhoA may be a key mediator of Ca2+ sensitization in response to G protein-coupled receptor activation (3, 13, 29). ROK phosphorylates the regulatory subunit of MLCP and inhibits its catalytic activity, thus resulting in increased myosin light chain phosphorylation, Ca2+ sensitization, and increased tension (29). Y-27632, a selective ROK inhibitor (32), effectively decreases Ca2+ sensitivity induced by various agonists such as carbachol, histamine, phenylephrine, endothelin-1, and U-46619 (3, 4, 13, 17, 32, 38). In the present study, Y-27632 caused a rightward shift in the [Ca2+]i-tension relationship in U-46619-pretreated pulmonary venous strips, which supports the concept that the ROK-associated pathway is involved in U-46619-induced Ca2+ sensitization in canine pulmonary veins.
TK inhibition attenuated the contractile response to lower concentrations of U-46619, but not the response to higher concentrations (Fig. 4). TK inhibition has recently been reported to attenuate U-46619 contraction in canine pulmonary veins (13), as well as hypoxic contraction in sheep pulmonary veins (33). TK has been reported to mediate agonist-induced increases in myofilament Ca2+ sensitivity in some vascular tissues (4, 13, 23, 28). However, we did not expect to see any effect of TK inhibition on the U-46619 [Ca2+]i-tension relationship, because the concentration of U-46619 used in the pulmonary venous strip studies (0.1 μM) was in the range where TK inhibition did not attenuate contraction in the ring studies. Surprisingly, we observed that TK inhibition caused a leftward shift in the U-46619 [Ca2+]i-tension relationship. This result reflects the fact that [Ca2+]i increased to lower values (P = 0.011) in U-46619 plus tyrphostin A-47-treated strips compared with U-46619-treated strips alone when extracellular Ca2+ was incrementally increased, whereas concomitant increases in tension were not significantly different (P = 0.371). TK inhibition has been reported to inhibit Ca2+ influx in bovine aorta (20) and to inhibit capacitative Ca2+ entry in rat ileal smooth muscle (24). In preliminary studies, we observed that TK inhibition inhibited capacitative Ca2+ entry in pulmonary veins (27). Thus it is possible that the leftward shift in the U-46619 [Ca2+]i-tension relationship was due to TK inhibition of Ca2+ influx and/or capacitative Ca2+ entry.
As noted earlier, only one study in the literature has assessed intracellular mechanisms that mediate U-46619 contraction in pulmonary veins (13). That study measured U-46619-induced changes in tension in endothelium-intact pulmonary venous rings and separately measured [Ca2+]i in dispersed pulmonary venous smooth muscle cells. Although there is some agreement between our results and results from this previous study (e.g., ROK and TK inhibition attenuated U-46619 contraction), there are also several significant differences. Perhaps the most striking difference involves U-46619-induced changes in [Ca2+]i. We observed large (similar to increases induced by 40 mM KCl) concomitant increases in [Ca2+]i and tension in every pulmonary venous smooth muscle strip that was studied. In contrast, the previous study reported that U-46619 (0.1 μM: same concentration as present study) did not evoke any changes in [Ca2+]i in 9 of 16 pulmonary venous smooth muscle cells tested, with the remaining cells exhibiting variable fluorometric responses to U-46619. We observed that administering nifedipine (10 μM) and removing extracellular Ca2+ attenuated U-46619-induced increases in tension and [Ca2+]i, whereas administering nifedipine (1 μM) and removing extracellular Ca2+ had no effect on U-46619 contraction in the previous study. We observed that inhibition of IP3 receptor-mediated Ca2+ release from the sarcoplasmic reticulum (2-APB) attenuated U-46619 contraction and the early peak increase in [Ca2+]i, whereas depletion of intracellular Ca2+ stores (cyclopiazonic acid) had no effect on U-46619 contraction in the previous study. Finally, we observed that PKC inhibition with BIS1 (3 μM) attenuated U-46619 contraction and caused a rightward shift in the U-46619 [Ca2+]i-tension relationship, whereas PKC inhibition with calphostin C or chelerythrine (both 1 μM) had no effect on U-46619 contraction in the previous study.
We can only speculate about the reasons responsible for these differences. We intentionally removed the endothelium in all protocols so that we could investigate the specific effects of U-46619 on pulmonary venous smooth muscle. The endothelium was not removed in the previous study, so U-46619-induced changes in tension include any modulating influence of the endothelium. In preliminary studies, we have observed that removing the endothelium potentiates U-46619 contraction (data not presented). We simultaneously measured changes in tension and [Ca2+]i in response to U-46619. The failure to see consistent changes in [Ca2+]i in response to U-46619 in the previous study could be a consequence of the cell isolation technique, although this seems unlikely because the cells exhibited substantial responses to caffeine (data not shown). We used higher concentrations of nifedipine than the previous study and a different PKC inhibitor, although we used the same concentration of nifedipine and the same PKC inhibitor in a recent study of pulmonary veins (4). We also used a higher concentration (3 μM vs. 1 μM) of the PKC inhibitor than used in the previous study. Finally, it should be noted that we investigated possible changes in myofilament Ca2+ sensitivity by assessing shifts in the U-46619 [Ca2+]i-tension relationship. In the previous study, changes in Ca2+ sensitivity were only inferred on the basis of the observation that U-46619 increased tension without consistent increases in [Ca2+]i.
There was some variability in the response to U-46619 under control conditions. This was most likely due to differences in vascular reactivity to U-46619 among dogs. To minimize this effect, we used paired pulmonary venous rings or strips from the same dog for control and experimental responses in each protocol.
In conclusion, U-46619 contractions in pulmonary veins are associated with increases in [Ca2+]i and myofilament Ca2+ sensitivity. U-46619 contraction is partially due to Ca2+ influx and Ca2+ release. The PKC, ROK, and TK signaling pathways all play a role in U-46619 contraction. In particular, the PKC and ROK pathways contribute to the U-46619-induced increase in myofilament Ca2+ sensitivity.
This work was funded by National Heart, Lung, and Blood Institute Grant HL-38291-18 and by Ohio Valley Affiliate of the American Heart Association Postdoctoral Fellowship 0425317B.
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