Regulation of pulmonary venous tone in response to muscarinic receptor activation

Xueqin Ding and Paul A. Murray

Center for Anesthesiology Research, The Cleveland Clinic Foundation, Cleveland, Ohio

Submitted 22 June 2004 ; accepted in final form 16 September 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We investigated cellular mechanisms that mediate or modulate the vascular response to muscarinic receptor activation (ACh) in pulmonary veins (PV). Isometric tension was measured in isolated canine PV rings with endothelium (E+) and without endothelium (E–). Tension and intracellular Ca2+ concentration ([Ca2+]i) were measured simultaneously in fura-2-loaded E– PV strips. In the absence of preconstriction, ACh (0.01–10 µM) caused dose-dependent contraction in E+ and E– rings. ACh contraction was potentiated by removing the endothelium or by nitric oxide (NO) synthase inhibition (N-nitro-L-arginine methyl ester, P = 0.001). Cyclooxygenase inhibition (indomethacin) reduced ACh contraction in both E+ and E– PV rings (P = 0.013 and P = 0.037, respectively). ACh contraction was attenuated by inhibitors of voltage-operated Ca2+ channels (nifedipine, P < 0.001), inositol-1,4,5-trisphosphate (IP3)-mediated Ca2+ release (2-aminoethoxydiphenyl borate, P = 0.001), PKC (bisindolylmaleimide I, P = 0.001), Rho-kinase (Y-27632, P = 0.002), and tyrosine kinase (TK; tyrphostin 47, P = 0.015) in E– PV rings. ACh (1 µM) caused a leftward shift in the [Ca2+]i-tension relationship (P = 0.015), i.e., ACh increased myofilament Ca2+ sensitivity. Inhibition of PKC, Rho-kinase, and TK attenuated the ACh-induced increase in myofilament Ca2+ sensitivity (P < 0.001, P < 0.001, and P = 0.024, respectively). These findings indicate that in canine PV, ACh contraction is modulated by NO and partially mediated by metabolites of the cyclooxygenase pathway and involves Ca2+ influx through voltage-operated Ca2+ channels and IP3-mediated Ca2+ release. In addition, ACh induces increased myofilament Ca2+ sensitivity, which requires the PKC, Rho-kinase, and TK pathways.

endothelium; nitric oxide; myofilament calcium sensitivity; calcium influx and release


INTEREST IN PULMONARY VEINS (PV) has increased recently following the report that a number of patients with atrial fibrillation have an ectopic electrical focus originating within the PV (31). PV are a primary site for entry of vagal nerves into the left atrium (73), although mechanisms of atrial fibrillation are clearly multifactorial (70). Pulmonary venous constriction may be involved in pulmonary edema formation in congestive heart failure (9), as well as in high altitude pulmonary edema (40). PV are known to constrict in response to a number of stimuli (8, 24, 51, 76).

The parasympathetic neurotransmitter, acetylcholine (ACh), is a muscarinic receptor agonist that has been reported to cause both PV relaxation (27) as well as PV contraction (30, 58, 62, 67). The PV response to ACh appears to depend on the concentration of ACh, the level of vasomotor tone, the muscarinic receptor subtype (M1–M5) and the species studied. ACh-induced PV relaxation appears to be mediated by endothelium-derived nitric oxide (NO), because it is abolished by removing the endothelium or by inhibiting NO synthase (43). However, the cellular mechanisms of ACh-induced contraction in PV have not been elucidated. In preliminary studies (65), we observed that ACh caused relaxation at lower concentrations (<1 µM) and contraction at higher concentrations in precontracted endothelium-intact (E+) canine PV, whereas only contraction was observed in endothelium-denuded (E–) PV. In addition, we reported that ACh contraction was mediated primarily by M3 muscarinic receptors in canine PV (65).

Contraction of vascular smooth muscle (VSM) is initiated by an increase in intracellular Ca2+ concentration ([Ca2+]i). This results from an influx of Ca2+ across the sarcolemma through plasma membrane channels [e.g., voltage-operated Ca2+ channels (VOCCs)], as well as Ca2+ release from the sarcoplasmic reticulum [e.g., inositol-1,4,5-trisphosphate (IP3)-mediated Ca2+ release]. However, VSM contraction is not simply proportional to changes in [Ca2+]i, because Ca2+ sensitivity of the contractile apparatus is another important mechanism for VSM contraction (61). Agonist-induced Ca2+ sensitization appears to be a G protein-mediated effect (39) and involves downstream effectors such as myosin light chain phosphatase (MLCP) (28), protein kinase C (PKC) (35), Rho-kinase (ROK) (64), and tyrosine kinases (TK) (18, 68). Furthermore, the endothelium plays a crucial role in regulating the tone of VSM via the release of vasoactive mediators (25).

Our goal was to investigate cellular mechanisms that either modulate or mediate the response of PV to ACh. Specifically, we investigated the role of the endothelium (NO and cyclooxygenase pathways), Ca2+ influx through VOCCs, and IP3-mediated Ca2+ release in the contractile response to ACh. In addition, we assessed the role of Ca2+ sensitization in the contractile response to ACh. The roles of the PKC, ROK, and TK signaling pathways were also investigated. To investigate the specific cellular mechanisms involved in ACh contraction, all experiments were performed under baseline tone conditions to avoid any effects of precontracting agonists.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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 adult 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 dog was 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 PV [third generation, 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. PV were cleaned of connective tissue and cut into ring segments 4–5 mm in length, with special care taken not to damage the endothelium. In some rings, the endothelium was denuded by gently rubbing the intimal surface with a cotton swab.

Isometric tension experiments. PV 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 PV 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 (i.e., no precontraction), a concentration-response curve to ACh was performed in each ring. This was achieved by increasing the concentration of ACh in half-log increments (from 0.01 µM to 10 µM) after the response to each preceding concentration had reached a steady state. The integrity of the endothelium was verified by assessing the vasorelaxant response to the endothelium-dependent vasodilator bradykinin (0.01 µM) during ACh contraction. Bradykinin caused ~25% relaxation in E+ PV rings, and this response was abolished in E– PV rings.

To assess the role of the endothelium in the contractile response to ACh, concentration-response curves to ACh were compared in E+ and E– PV rings. To investigate the role of NO in modulating ACh contraction, E+ PV rings were pretreated for 30 min with N-nitro-L-arginine methyl ester (L-NAME, 100 µM), an inhibitor of NO synthase. To investigate the role of cyclooxygenase metabolites in ACh contraction, E+ and E– PV rings were pretreated for 30 min with indomethacin (10 µM), a cyclooxygenase inhibitor. The contractile responses to ACh in L-NAME- and indomethacin-pretreated rings were compared with responses in untreated paired rings.

To characterize the relative contributions of Ca2+ influx and Ca2+ release to the total contractile response to ACh in PV smooth muscle, E– PV rings were pretreated with nifedipine (10 µM), a VOCC inhibitor, and 2-aminoethoxydiphenyl borate (2-APB, 100 µM), an IP3-mediated Ca2+ release inhibitor. After incubation with these inhibitors for 30 min, the rings were contracted with ACh (0.01–10 µM). The contractile responses to ACh in nifedipine- and 2-APB-pretreated rings were compared with responses in untreated paired rings.

To identify the signaling pathways involved in the contractile response to ACh, PV rings were incubated with the following inhibitors, alone or in combination: bisindolylmaleimide I (BIS1, 3 µM), a PKC inhibitor; Y-27632 (1 µM), a ROK inhibitor; and tyrphostin 47 (100 µM), a TK inhibitor. The concentrations of inhibitors in the present study are within the range of concentrations used in previous studies (14, 33, 56, 66). PV rings were pretreated with these inhibitors for 30 min before ACh contraction. None of the inhibitors had an effect on baseline tension.

Preparation of pulmonary venous smooth muscle strips. Intralobar PV (2–4 mm ID) were dissected carefully and immersed in cold modified KRB solution. The PV were cleaned of connective tissue and cut into strips (2x6 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 intracellular Ca2+ concentration. Intralobar PV strips without endothelium were loaded with 5 µM acetoxylmethyl ester of fura-2 (fura-2 AM) solution. A noncytotoxic detergent, 0.05% cremophor EL, was added to solubilize the fura-2 AM in the solution. After fura-2 loading, the PV 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 (Grass FTO3; Grass Instrument, Quincy, MA) 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 using 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 concentration 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 also measured in all experiments, and the signals were observed to change in opposite directions in response to the various interventions. Because each PV strip served as its own control, background fluorescence was assumed to be constant and was not subtracted from the calculated 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 collected with a software package from Photon Technology International.

Myofilament Ca2+ sensitivity experiments. We investigated the effects of ACh on the [Ca2+]i-tension relationship. In each PV 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 ACh-pretreated (1 µM) strips in an incremental fashion from 0 to 0.125, 0.25, 0.5, 1.25, and 2.5 mM. Changes in tension and [Ca2+]i are expressed as a percentage of the response to the first application of 40 mM KCl. The same procedure was repeated in strips pretreated with BIS1 (3 µM), Y-27632 (1 µM), or tyrphostin 47 (100 µM).

Solutions and chemicals. Drugs of the highest purity commercially available were utilized: ACh, L-NAME, indomethacin, nifedipine, 2-APB, BIS1, cremophor EL, dimethyl sulfoxide (Sigma, St. Louis, MO); tyrphostin 47, Y-27632 (Calbiochem-Novabiochem, San Diego, CA); and fura-2 AM (Texas Fluorescence Labs, Austin, TX). BIS1, tyrphostin 47, 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.

Data analysis. All data are expressed as means ± SD. Contractile responses to ACh in the ring studies are expressed as the percentage contraction induced by 60 mM KCl. The ACh contractile responses were compared in matched control and "treated" rings or strips from the same dogs. Two-way analysis of variance for repeated measures followed by contrast analysis and Bonferroni correction were used for comparisons within and between groups. For rings studies, ACh dose was used as the within-subject factor and treatment (with or without) was used as the between-subject factor. For strips studies, Ca2+ concentration was used as the within-subject factor and treatment (with or without) was used as the between-subject factor. All statistical analyses utilized SPSS for WINDOWS software (version 11.5; SPSS, Chicago, IL). A P value of <0.05 was chosen as significant. In all experiments, sample size (n values) equals the number of dogs from which PV rings or strips were taken.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Role of the endothelium in ACh contraction. We tested the hypothesis that NO modulates ACh contraction in PV. ACh caused dose-dependent (P < 0.001) contraction in both E+ and E– PV rings (Fig. 1A). ACh contraction in the E– group was increased (P < 0.001) compared with the E+ group (Fig. 1A). Likewise, ACh contraction was potentiated (P = 0.001) after treatment with L-NAME (Fig. 1B). These results indicate that endothelium-derived NO acts to modulate ACh contraction in PV.



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Fig. 1. A: effect of removing the endothelium on ACh contraction in isolated canine pulmonary veins (PV). B: effect of nitric oxide (NO) synthase inhibition [N-nitro-L-arginine (L-NAME) 100 µM] on ACh contraction in endothelium-intact (E+) PV. ACh contraction was potentiated by removing the endothelium (P < 0.001) and by NO synthase inhibition (P = 0.001). E–, endothelium denuded; error bars represent SD in all figures; n = 6.

 
Role of the cyclooxygenase pathway in ACh contraction. We tested the hypothesis that vasoconstrictor metabolites of the cyclooxygenase pathway mediate ACh contraction in PV. The cyclooxygenase inhibitor indomethacin had no effect on resting tension. Pretreatment of E+ rings with indomethacin decreased (P = 0.013) ACh contraction (Fig. 2A). To determine whether this was an endothelial or smooth muscle effect, we also assessed ACh contraction in E– PV pretreated with indomethacin. Under these conditions, ACh contraction was also attenuated (P = 0.037) (Fig. 2B). These results suggest that a component of ACh contraction is mediated by cyclooxygenase metabolites primarily released from pulmonary venous smooth muscle (PVSM).



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Fig. 2. A: effect of cyclooxygenase inhibition (10 µM indomethacin) on ACh contraction in E+ PV. B: effect of indomethacin (10 µM) on ACh contraction in E– PV. Indomethacin attenuated ACh contraction in both E+ (P = 0.013) and E– (P = 0.037) PV (n = 6).

 
Roles of VOCCs and IP3-mediated Ca2+ release in ACh contraction. We tested the hypothesis that transarcolemmal Ca2+ influx via VOCCs and/or IP3-mediated Ca2+ release from the sarcoplasmic reticulum are involved in ACh contraction in PV. Neither inhibition of VOCCs (nifedipine) nor inhibition of IP3 receptors (2-APB) had an effect on resting tension in E– PV. However, ACh contraction was attenuated (P < 0.001) after pretreatment with nifedipine (Fig. 3A) and 2-APB (Fig. 3B). These results indicate that both Ca2+ influx via VOCCs and Ca2+ release via IP3 receptor activation mediate a component of ACh contraction.



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Fig. 3. A: effect of voltage-operated Ca2+ channel (VOCC) inhibition (nifedipine 10 µM) on ACh contraction in E– PV. B: effect of IP3-mediated Ca2+ release inhibition [2-aminoethoxydiphenyl borate (2-APB) 100 µM] on ACh contraction in E– PV. ACh contraction was attenuated by inhibition of VOCCs (P < 0.001) and inositol-1,4,5-trisphosphate (IP3)-mediated Ca2+ release (P < 0.001, n = 6).

 
Roles of the PKC, ROK, and TK signaling pathways in ACh contraction. We tested the hypothesis that ACh contraction in PV involves the PKC, ROK, or TK signaling pathways. PKC inhibition (BIS1), ROK inhibition (Y-27632), and TK inhibition (tyrphostin 47) had no effect on resting tension in E– PV. However, PKC inhibition (P < 0.001), ROK inhibition (P = 0.02), and TK inhibition (P = 0.015) each attenuated ACh contraction (Fig. 4, A, B, and C, respectively). Combined treatment with all three inhibitors essentially abolished (P < 0.001) ACh contraction (Fig. 5). These results indicate that the PKC, ROK, and TK signaling pathways are involved in ACh contraction in PV.



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Fig. 4. A: effect of protein kinase C (PKC) inhibition [bisindolylmaleimide I (BIS1) 3 µM] on ACh contraction in E– PV. B: effect of Rho-kinase (ROK) inhibition (1 µM Y-27632) on ACh contraction in E– PV. C: effect of tyrosine kinase (TK) inhibition (100 µM tyrphostin 47) on ACh contraction in E– PV. Inhibition of PKC (P < 0.001), ROK (P = 0.02), and TK (P = 0.015) attenuated ACh contraction (n = 6).

 


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Fig. 5. Effect of combined inhibition of PKC, ROK, and TK (3 µM BIS1 + 1 µM Y-27632 + 100 µM tyrphostin 47) on ACh contraction in E– PV. Inhibition of these 3 signaling pathways essentially abolished ACh contraction (P < 0.001, n = 4).

 
Role of myofilament Ca2+ sensitivity in ACh contraction. We tested the hypothesis that ACh increases myofilament Ca2+ sensitivity in PV. Control and ACh pretreated E– PV strips in a Ca2+-free buffer containing 40 mM KCl were exposed to incremental increases in extracellular Ca2+ concentration. The [Ca2+]i-tension relationships in control and ACh-pretreated PV strips are summarized in Fig. 6. Compared with control, ACh caused a leftward shift (P = 0.015) in the [Ca2+]i-tension relationship. Thus for a given value of [Ca2+]i, tension was greater in ACh-pretreated strips compared with control (i.e., ACh increased myofilament Ca2+ sensitivity).



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Fig. 6. Summarized data for the effects of ACh on the intracellular Ca2+ concentration ([Ca2+]i)-tension relationship. Control and ACh-pretreated pulmonary venous smooth muscle (PVSM) strips were bathed in a Ca2+-free buffer containing 40 mM KCl. We increased extracellular Ca2+ incrementally from 0 to 2.5 mM while simultaneously measuring [Ca2+]i and tension. ACh (1 µM) caused a leftward shift (P = 0.015) in the [Ca2+]i-tension relationship (n = 6).

 
Role of PKC, ROK, and TK signaling pathways in ACh-induced increase in myofilament Ca2+ sensitivity. We tested the hypothesis that the PKC, ROK, and TK pathways mediate the ACh-induced increase in myofilament Ca2+ sensitivity. PV strips were pretreated with BIS1, Y-27632, or tyrphostin 47 to inhibit the respective signaling pathways. Experiments were then performed as described above to generate [Ca2+]i-tension relationships in ACh-pretreated PV with or without the signaling pathway inhibitors. As summarized in Fig. 7, myofilament Ca2+ sensitivity in ACh-pretreated PV strips was attenuated by inhibition of PKC (P < 0.001) (Fig. 7A), ROK (P < 0.001) (Fig. 7B) and TK (P = 0.024) (Fig. 7C).



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Fig. 7. A: effect of PKC inhibition (3 µM BIS1) on the ACh-induced change in the [Ca2+]i-tension relationship. B: effect of ROK inhibition (1 µM Y-27632) on the ACh-induced change in the [Ca2+]i-tension relationship. C: effect of TK inhibition (100 µM tyrphostin 47) on the ACh-induced change in the [Ca2+]i-tension relationship. The ACh-induced change in the [Ca2+]i-tension relationship was attenuated by inhibition of PKC (P < 0.001), ROK (P < 0.001), and TK (P = 0.024; n = 6).

 

    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Our results indicate that in canine PV, removing the endothelium or NO synthase inhibition potentiated, whereas cyclooxygenase inhibition attenuated, ACh-induced contraction. Inhibition of Ca2+ influx or Ca2+ release, as well as inhibition of the PKC, ROK, and TK signaling pathways, attenuated ACh-induced contraction. ACh caused a leftward shift in the [Ca2+]i-tension relationship. Inhibition of PKC, ROK, and TK attenuated the ACh-induced increase in the [Ca2+]i-tension relationship. A "cartoon" summarizing these cellular mechanisms mediating the effects of ACh on PV is presented in Fig. 8.



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Fig. 8. Summarized "cartoon" for the signal transduction pathways involved in muscarinic receptor activation by ACh in canine PV. M, muscarinic; EC, endothelial cells; SR, sarcoplasmic reticulum; AA, arachidonic acid; PLA2, phospholipase A2; PLC, phospholipase C; DAG, diacylglycerol; PGI2, prostacyclin; TXA2, thromboxane A2; sGC, soluble guanylate cyclase; cGMP, cyclic guanosine monophosphate (cGMP); COX, cyclooxygenase; MLCP, myosin light chain phosphatase; MLC20, regulatory myosin light chain; P, phosphorylation. The solid lines represent the signaling pathways that regulate ACh contraction. The dashed lines represent the signaling pathways that mediate the ACh-induced increase in myofilament Ca2+ sensitivity.

 
Role of endothelium-derived NO. It is known that ACh can cause vasoconstriction by directly stimulating muscarinic receptors on VSM (35) and by activating muscarinic receptors on the vascular endothelium to release endothelium-derived contracting factors (42). The vascular endothelium also plays a significant modulating role in the control of VSM tone by producing endothelium-derived relaxing factors (EDRFs). NO is a major EDRF, and ACh is capable of triggering the endothelium to release NO through muscarinic receptor stimulation (11). NO release from endothelial cells causes vasodilation in nearby VSM cells by stimulating guanylyl cyclase and thereby elevating the intracellular concentration of cyclic guanosine monophosphate. Previous studies (62, 67) reported that ACh caused contraction in ovine PV at resting tension. Removing the endothelium and NO synthase inhibition augmented ACh contraction in PV. In agreement with these studies, we also observed that ACh causes contraction in both E+ and E– rings at resting tension. Removing the endothelium or pretreatment with L-NAME significantly augmented PV contraction to ACh. Thus NO appears to modulate ACh-induced contraction in PV. It has been reported that ACh increases pulmonary vascular resistance by contracting the venous segments of the canine pulmonary circulation (20) and to induce pulmonary edema (15). Therefore, NO may protect the lung by modulating the PV contractile response to ACh.

Role of the cyclooxygenase pathway. Endothelium and VSM are capable of producing a variety of vasoactive substances that are products of arachidonic acid metabolism via the cyclooxygenase pathway. Among these are prostacyclin and thromboxane, which cause vasodilation and vasoconstriction, respectively. It has been reported that ACh contraction is partially mediated by contractile cyclooxygenase products in isolated rat heart (5) and isolated porcine coronary epicardial arteries (44). Contractile cyclooxygenase metabolites can inhibit voltage-gated K+ channels, leading to depolarization, activation of L-type Ca2+ channels, and vasoconstriction (13). In addition to Ca2+ influx, Ca2+ release and myofilament Ca2+ sensitivity are also involved in thromboxane-induced contraction in canine PV (34). In the present study, we used 10 µM indomethacin to investigate the effect of inhibiting the cyclooxygenase pathway on ACh contraction. This concentration of indomethacin has been used by us (57, 59) and by other investigators (19, 26, 32). Moreover, higher concentrations of indomethacin (100 and 250 µM) have been reported to cause a Ca2+ antagonist effect (46, 47). Our results showed that cyclooxygenase inhibition attenuated ACh contraction in both E+ and E– PV rings, which indicates that ACh primarily stimulates release of constrictor prostaglandins from PVSM. Constrictor prostaglandins may mediate a relatively small component (30%) of ACh-induced PV contraction by increasing Ca2+ mobilization or Ca2+ sensitivity.

Role of Ca2+ influx and Ca2+ release. Muscarinic receptors transduce their signals by coupling with G proteins. ACh binds to muscarinic receptors coupled to Gq and activates phospholipase C-mediated hydrolysis of phosphatidylinositol-bis-phosphate to IP3 and diacylglycerol, which causes Ca2+ release and PKC activation, respectively. Recently, it has been reported that ACh induced Ca2+ release from internal stores by activating muscarinic receptors in mudpuppy taste cells (48) and cat detrusor muscle cells (3). Consistent with these reports, we demonstrated that ACh contraction is mediated by IP3-dependent Ca2+ release in PVSM. In addition to Ca2+ release, our results showed that pretreatment with nifedipine attenuated ACh contraction in PV, which suggests that ACh contraction also involves Ca2+ influx through VOCCs. Chau et al. (10) and Kawano et al. (37) also reported that ACh contraction involves Ca2+ influx in rat pulmonary artery and human mesenchymal stem cells, respectively. The mechanism by which ACh opens VOCCs is still unknown. However, ACh has been reported in rat pancreatic {beta}-cells to cause depolarization, open VOCCs, and allow Ca2+ influx (45). Whether ACh induces Ca2+ influx through VOCCs by depolarization in PVSM remains to be elucidated.

Role of PKC, ROK, and TK in ACh contraction. A number of investigations have concluded that PKC, ROK, and TK contribute to smooth muscle contraction by regulating Ca2+ (17, 36, 74, 75) or myofilament Ca2+ sensitivity (2, 38, 41, 54). Therefore, we investigated the effect of PKC, ROK, and TK inhibition on ACh contraction in canine PV. Our results show that inhibition of PKC, ROK, or TK alone attenuated ACh contraction, and combined inhibition of these signaling pathways essentially abolished ACh contraction. These results indicate that all three signaling pathways are involved in ACh contraction. We next assessed the role of these signaling pathways in the ACh-induced increase in myofilament Ca2+ sensitivity.

Myofilament Ca2+ sensitivity. An increase in cytoplasmic [Ca2+]i is generally considered the key event for the activation process of the VSM contractile apparatus. However, the relation linking [Ca2+]i to the force of contraction represents the Ca2+ sensitivity of the contractile apparatus. Ca2+ sensitivity can be modified by the action of agonists or drugs, as well as in some pathophysiological conditions (55). The major mechanism of Ca2+ sensitization of smooth muscle contraction is through inhibition of the smooth muscle MLCP, which increases regulatory myosin light chain (MLC20) phosphorylation (55). Two molecular mechanisms of MLCP inhibition have been proposed (50). First, receptor-mediated activation of the small G protein RhoA leads to activation of ROK, which inhibits MLCP through phosphorylation of its regulatory subunit either directly or via ZIP-like kinase (2). Second, PKC isoforms have been identified to phosphorylate CPI-17, which, in the phosphorylated state, inhibits MLCP specifically (21, 22). In addition, TK phosphorylation has been suggested to play an important role in Ca2+ sensitization by directly increasing MLC20 phosphorylation (16).

It has been reported that muscarinic receptor agonists activate a GTP-dependent messenger cascade that increases myofilament Ca2+ sensitivity (30). Although the mechanism of this action is not fully known, there is considerable evidence that the Ca2+- and lipid-dependent enzyme PKC plays a key role (60, 69). As noted above, ACh binds to muscarinic receptors coupled to Gq and activates PKC. The contribution of PKC to Ca2+ sensitization induced by muscarinic receptor stimulation seems to be species, preparation, and muscarinic receptor subtype dependent. In canine tracheal smooth muscle, Bremerich et al. (7) found no evidence that PKC mediates increases in Ca2+ sensitivity produced by muscarinic receptor stimulation. Similar results were reported in guinea pig stomach smooth muscle (49). However, PKC activation is involved in Ca2+ sensitization induced by muscarinic receptor activation in smooth muscle from rabbit aorta, rabbit bladder, and human bladder (77). In the current study of canine PVSM, muscarinic receptor stimulation with ACh increased myofilament Ca2+ sensitivity. The increase in myofilament Ca2+ sensitivity induced by ACh was attenuated by inhibition of PKC. However, PKC inhibition did not abolish the ACh-induced increase in myofilament Ca2+ sensitivity, implying that other signaling pathways may be involved.

Similar to PKC, the contribution of ROK to Ca2+ sensitization induced by muscarinic receptor stimulation in smooth muscle seems to be species, preparation, and muscarinic receptor subtype dependent. In rabbit stomach fundus smooth muscle (52) and chicken gizzard smooth muscle (4), ROK is reported to be present but to play no role in muscarinic receptor-induced contraction. However, ROK-mediated Ca2+ sensitization contributes to contraction induced by muscarinic receptor activation in mouse anococygeus and rat bronchial smooth muscle (6, 12). In the present study of canine PVSM, ROK inhibition attenuated the ACh-induced increase in myofilament Ca2+ sensitivity, which suggests that this signaling pathway plays a role.

TK phosphorylation is another important mechanism for regulation of smooth muscle contraction and Ca2+ sensitivity (16). It has been reported that three different TK inhibitors reduced the amplitude of contraction of intact smooth muscle stimulated by muscarinic agonists (18). At least two sites between receptor activation of smooth muscle and contraction may be subject to regulation by protein tyrosine phosphorylation. The first includes mechanisms that couple receptor activation to increases in cytosolic Ca2+ resulting from enhanced influx of extracellular Ca2+ and/or release of intracellular Ca2+ from vesicular storage sites (17, 75). The second site of action for protein tyrosine phosphorylation may involve mechanisms that control agonist-induced increases in Ca2+ sensitivity of the contractile apparatus (63). Steusloff and coworkers (63) found an apparent dissociation between TK inhibitor (genistein)-mediated suppression of Ca2+ sensitivity and changes in MLC20 phosphorylation. However, in their study, high levels of MLC20 phosphorylation (20%) persisted after incubation with genistein, even though force decreased by >90%. This result suggests that mechanisms in addition to modulation of MLC20 phosphorylation participate in regulating Ca2+ sensitivity. In the present study, TK inhibition attenuated ACh contraction and the ACh-induced increase in myofilament Ca2+ sensitivity, which suggests that TK also plays a role.

The response to ACh has been investigated in human PV (71, 72). After precontraction with norepinephrine, ACh caused dose-dependent relaxation of human PV, which is similar to our previous study in precontracted canine PV (65). However, in contrast to the results of the present study, Walch and coworkers observed either a modest relaxation (71) or no effect (72) in response to ACh under conditions of resting tone. It is possible that ACh contraction does not occur in human PV. However, it is important to note that all human PV studied by Walch and coworkers were obtained from lung lobes surgically removed because of cancer. It is conceivable that cancer (or perhaps chemotherapy) may have altered the pulmonary vasculature. For example, it is well established that an increase in NO production is associated with a number of lung diseases, including cancer. The development of lung cancer depends on an adequate vascular supply to the tumor, so a variety of mechanisms (cytokines, growth factors, angiogenesis factors) could have chronically altered PV regulation in the lungs utilized by Walch and coworkers.

There was some variability in the response to ACh under control conditions. This was most likely due to differences in vascular reactivity to ACh among dogs. To minimize this effect, we used paired segments from the same dog for control and experimental responses in each protocol.

In the present study, we used bradykinin to verify the integrity of the endothelium because bradykinin-induced endothelium-dependent relaxation has been reported in canine (1), porcine (23), ovine (67), and bovine PV (29). Sai and coworkers (53) reported that bradykinin induced endothelium-independent relaxation in canine PV. In contrast, we observed that bradykinin relaxation was abolished in E– PV. Differences in dog size (large vs. small) or rings used (paired vs. not paired) for E+ and E– groups could contribute to the difference. In addition, our results are consistent with those of Aarnio et al. (1) in canine PV.

In summary. Under baseline tone conditions, ACh caused contraction in both E+ and E– canine PV. NO modulates ACh contraction in E+ PV. The contractile response to ACh is at least partially mediated by cyclooxygenase metabolites. In canine PVSM, the contractile response to ACh is due to Ca2+ influx through voltage-operated Ca2+ channels, IP3-mediated Ca2+ release, as well as an increase in myofilament Ca2+ sensitivity, and involves activation of the PKC, ROK, and TK signaling pathways.


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This work was funded by National Heart, Lung, and Blood Institute Grant HL-38291-17 and by Ohio Valley Affiliate of the American Heart Association Postdoctoral Fellowship 0425317B.


    FOOTNOTES
 

Address for reprint requests and other correspondence: P. A. Murray, Center for Anesthesiology Research, FF40, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195 (E-mail: murrayp{at}ccf.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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