Role of PKC, tyrosine kinases, and Rho kinase in alpha -adrenoreceptor-mediated PASM contraction

Derek S. Damron, Noriaki Kanaya, Yasuyuki Homma, Si-Oh Kim, and Paul A. Murray

Center for Anesthesiology Research, Division of Anesthesiology and Critical Care Medicine, Cleveland Clinic Foundation, Cleveland, Ohio 44195


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our objectives were to identify the relative contributions of intracellular free Ca2+ concentration ([Ca2+]i) and myofilament Ca2+ sensitivity in the pulmonary artery smooth muscle (PASM) contractile response to the alpha -adrenoreceptor agonist phenylephrine (PE) and to assess the role of PKC, tyrosine kinases (TK), and Rho kinase (ROK) in that response. Our hypothesis was that multiple signaling pathways are involved in the regulation of [Ca2+]i, myofilament Ca2+ sensitization, and vasomotor tone in response to alpha -adrenoreceptor stimulation of PASM. Simultaneous measurement of [Ca2+]i and isometric tension was performed in isolated canine pulmonary arterial strips loaded with fura 2-AM. PE-induced tension development was due to sarcolemmal Ca2+ influx, Ca2+ release from inositol 1,4,5-trisphosphate-dependent sarcoplasmic reticulum Ca2+ stores, and myofilament Ca2+ sensitization. Inhibition of either PKC or TK partially attenuated the sarcolemmal Ca2+ influx component and the myofilament Ca2+ sensitizing effect of PE. Combined inhibition of PKC and TK did not have an additive attenuating effect on PE-induced Ca2+ sensitization. ROK inhibition slightly decreased [Ca2+]i but completely inhibited myofilament Ca2+ sensitization. These results indicate that PKC and TK activation positively regulate sarcolemmal Ca2+ influx in response to alpha -adrenoreceptor stimulation in PASM but have relatively minor effects on myofilament Ca2+ sensitivity. ROK is the predominant pathway mediating PE-induced myofilament Ca2+ sensitization.

intracellular calcium ion; myofilament calcium ion sensitivity; phenylephrine; vascular smooth muscle; pulmonary artery smooth muscle; protein kinase C


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CATECHOLAMINE-INDUCED ACTIVATION of alpha -adrenoreceptors on vascular smooth muscle cells results in an increase in vasomotor tone. Vasomotor tone is regulated by intracellular free Ca2+ concentration ([Ca2+]i) and myofilament Ca2+ sensitivity. The initial increase in tone is mediated by an increase in [Ca2+]i, which triggers activation of myosin light chain kinase, myosin light chain phosphorylation, and an increase in tension. Maintenance of vascular smooth muscle contraction is more complex and likely involves several signaling pathways. Sensitization of the contractile apparatus to Ca2+ appears to be one important mechanism of pharmacomechanical coupling (12). Activation of PKC (4, 5, 11, 22), tyrosine kinases (TK) (7, 18, 19, 42), and/or Rho kinase (ROK) (18, 46) have been suggested to play important roles in the signal transduction events associated with vascular smooth muscle contraction. However, the relative roles of PKC, TK, and ROK in regulating [Ca2+]i, myofilament Ca2+ sensitivity, and tension in response to alpha -adrenoreceptor stimulation have not been fully elucidated.

Although the cellular mechanisms involved in alpha -adrenoreceptor-mediated vasoconstriction have been studied in a variety of systemic vascular smooth muscle types (14, 40, 45, 49), comparatively less is known about the cellular mechanisms mediating alpha -adrenoreceptor activation in the pulmonary circulation. Moreover, differential responses to hypoxia in the systemic (vasodilation) vs. the pulmonary (vasoconstriction) circulation underscore the difficulty in extrapolating results obtained in systemic vascular smooth muscle to cellular mechanisms that regulate pulmonary vasomotor tone. In the pulmonary circulation, the extent to which PKC, TK, and ROK mediate alpha -adrenoreceptor pulmonary vasoconstriction is controversial (5, 18, 19, 22). In endothelium-intact canine pulmonary artery smooth muscle (PASM) rings, norepinephrine-induced contractions were insensitive to PKC inhibitors but were abolished by TK inhibition and ROK inhibition (18). In contrast, others demonstrated that the vasoconstrictor response to norepinephrine in the feline pulmonary vascular bed was attenuated by PKC inhibitors (22) and likely mediated by Ca2+-independent PKC-delta isozyme/calmodulin-dependent kinase III (5). Others suggested that the contractile response to norepinephrine in isolated rat pulmonary artery is largely mediated by TK activation (42). In none of these studies were [Ca2+]i and tension simultaneously measured in the same tissue so that the role of PKC, TK, or ROK in [Ca2+]i and myofilament Ca2+ sensitivity in response to alpha -adrenoreceptor activation could be adequately assessed. Only one study assessed the role of sarcolemmal Ca2+ influx, as well as release of Ca2+ from the sarcoplasmic reticulum, in mediating the actions of alpha -adrenoreceptor activation in PASM (17). However, that study did not directly measure [Ca2+]i nor did it assess the role(s) of protein kinase activation in mediating increases in tension in response to alpha -adrenoreceptor activation (17). The present study is unique in that our goal was to simultaneously measure [Ca2+]i and tension in PASM strips and to identify the extent to which PKC, TK, and ROK modulate [Ca2+]i and myofilament Ca2+ sensitivity in response to alpha -adrenoreceptor stimulation with phenylephrine (PE).


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

All surgical procedures and experimental protocols were approved by the Cleveland Clinic Foundation Institutional Animal Care and Use Committee (Cleveland, OH).

Preparation of pulmonary arterial strips. Heartworm-negative mongrel dogs (20-30 kg) were anesthetized with intravenous pentobarbital sodium (30 mg/kg) and fentanyl citrate (15 µg/kg) and placed on positive-pressure ventilation. A catheter was inserted into 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 electrically induced ventricular fibrillation. The heart and lungs were removed from the thorax en bloc. Right and left intralobar pulmonary arteries [2- to 4-mm internal diameter (ID)] were dissected free and immersed in cold modified Krebs-Ringer bicarbonate (KRB) solution composed of (in mM) 118.3 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 2.5 CaCl2, 25 NaHCO3, 0.016 Ca-EDTA, and 11.1 glucose. The pulmonary arteries were cleaned of fat and connective tissue and cut into strips (2 × 8 mm) or rings (2- to 4-mm ID). The endothelium was denuded by gently rubbing the intimal surface with a cotton swab. The absence of an intact endothelium was later verified by assessing the vasorelaxant response to 10-6 M acetylcholine.

Simultaneous measurement of [Ca2+]i and tension. As described previously (37), pulmonary arterial strips without endothelium were loaded with 5 µM fura 2-AM at room temperature (22-24°C). A noncytotoxic detergent, 0.05% cremophor EL, was added to solubilize the fura 2-AM in the solution and act as a vehicle for penetrating the tissue. After fura 2 loading, the muscle strips were washed with KRB buffer for 30-60 min to remove unhydrolyzed fura 2-AM and then placed between two stainless steel hooks in a temperature-controlled (37°C) cuvette (volume 3 ml), which was continuously perfused (12 ml/min) with KRB solution bubbled with 95% air-5% CO2 (pH 7.4). One hook was anchored, and the other was connected to a strain gauge transducer (Grass model FTO3; Quincy, MA) to measure isometric force. The resting tension was adjusted to 4 g, which was determined in preliminary studies to be optimal for achieving a maximum contractile response to 20 mM KCl. 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. Fura 2 fluorescence signals (340 nm, 380 nm, and 340 nm-to-380 nm ratio) were continuously monitored at a sampling frequency of 2.5 Hz and collected with a software package from Photon Technology International. The 340-to-380 fluorescence ratio was used as an indicator of [Ca2+]i. In control experiments, the baseline fluorescence intensities, as well as the signal ratios measured in the absence of dye, were not altered by any of the experimental interventions. After changes in tension and [Ca2+]i in response to 60 mM KCl were measured, the strips were washed with fresh KRB for 30 min. All strips were pretreated with propranolol (5 µM, 30 min) to inhibit the beta -agonist effect of PE. A high dose of PE (10 µM) was used throughout the study to achieve a significant increase in tension in protocols in which extracellular Ca2+ was absent. The response to PE (10 µM) was assessed in the presence (10 min) or absence of the PKC inhibitor (48) bisindolylmaleimide I (Bis I; 3 µM), the TK inhibitor (26) tyrphostin (Tyr) A47 (10 µM), the ROK inhibitor (15) Y-27632 (10 µM), or the inositol 1,4,5-trisphosphate (IP3) receptor inhibitor (2) 2-aminoethoxydiphenyl borate (2-APB; 100 µM). Structurally different inhibitors of PKC (calphostin C; 0.1, 0.3, and 1 µM) and TK (genistein; 100 µM) were also investigated. Structurally similar inactive analogs of Bis (Bis V; 3 µM), Tyr A47 (Tyr A1; 10 µM) and genistein (daidzein, 100 µM) were also assessed. Nonspecific effects of the inhibitors on voltage-gated Ca2+ channels and myosin light chain kinase activity were assessed in strips contracted with KCl. Bis I, Bis V, calphostin C, Tyr A47, Tyr A1, genistein, daidzein, and 2-APB had no effect on KCl-induced contraction, whereas Y-27632 inhibited the increase in tension by 20 ± 9%. Early [Ca2+]i and tension values represent the peak increases in each parameter in the 2 min after administration of PE. Late [Ca2+]i and tension values represent measurements for each parameter 15 min after administration of PE. Summarized data for the changes in [Ca2+]i and tension after an intervention are expressed as the percent change from the previous response measured in that particular protocol.

Materials. Phenylephrine HCl, propranolol HCl, Tyr A47, Tyr A1, genistein, daidzein, Bis I, and Bis V were purchased from Sigma (St. Louis, MO). 2-APB and calphostin C were obtained from Calbiochem (La Jolla, CA). Y-27632 was obtained from Biomol (Plymouth Meeting, PA).

Statistical analysis and data presentation. Results are expressed as means ± SE. The sample size, n, represents the number of dogs from which strips or rings were studied. Statistical comparisons used ANOVA and the Bonferroni-Dunn post hoc test. Student's t-test for paired comparisons was used when appropriate. Differences were considered statistically significant at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of KCl and PE on [Ca2+]i and tension. Figure 1A illustrates simultaneous recordings of [Ca2+]i and tension during exposure to 60 mM KCl and 10 µM PE in a PASM strip. Addition of KCl resulted in rapid, early increases in [Ca2+]i and tension, followed by late, sustained increases near peak levels until washout. PE caused early peak increases in [Ca2+]i and tension followed by late, sustained increases at lower values. PE increased tension to a similar degree as KCl, whereas the increase in [Ca2+]i was only about half of that observed with KCl. During washout of PE, tension remained elevated even when [Ca2+]i had returned to baseline (Fig. 1A). PE caused a leftward shift in the continuous [Ca2+]i-tension relationship (Fig. 1B). The reproducibility of the PE response was assessed by administering PE three successive times to PASM strips. As summarized in Fig. 2, increases in [Ca2+]i and tension in response to the second and third applications of PE were similar although somewhat greater then the first response to PE. We next investigated the effects of the various interventions on the early (defined as the peak changes occurring within 2 min of PE application) and late (measured 15 min after administration of PE) changes in [Ca2+]i and tension in response to PE in PASM strips.


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 1.   A: changes in intracellular free Ca2+ concentration ([Ca2+]i) (indicated by 340 nm-to-380 nm ratio) and tension during contractions induced by 60 mM KCl and 10 µM phenylephrine (PE) in a canine pulmonary artery smooth muscle (PASM) strip. Extracellular Ca2+ concentration was 2.5 mM. Vertical bar represents the period after washout (w/o) of PE when tension remains increased above baseline whereas [Ca2+]i has returned to baseline. B: continuous changes in the [Ca2+]i-tension relationship in response to KCl and PE. Data presented in A are plotted as a phase-plane loop in B.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 2.   Summarized data depicting the reproducibility of early (measured within 2 min) and late (measured at 15 min) increases in [Ca2+]i (A) and tension (B) in response to 3 consecutive applications of PE. *Significantly different from first PE (PE-1) response (P < 0.05); n = 6.

Effect of removing extracellular Ca2+. To test the hypothesis that influx of extracellular Ca2+ is required for PE-induced contraction, we measured [Ca2+]i and tension in response to PE in the presence or absence of extracellular Ca2+. As illustrated in Fig. 3, removal of extracellular Ca2+ decreased the early increase in [Ca2+]i and tension by 32 ± 6% and 55 ± 5%, respectively. In the absence of extracellular Ca2+ the late phase of the PE-induced increase in [Ca2+]i was abolished, whereas the late phase of tension development was reduced by 65 ± 2%.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 3.   A: Changes in [Ca2+]i and tension induced by 10 µM PE in the presence (2.5 mM; Control) or absence of extracellular Ca2+ in a PASM strip. Note that the time scales for [Ca2+]i and tension are different. The original tracings for [Ca2+]i are designed to highlight the early response to PE. B: summarized data. *Significantly different from response measured in the presence of extracellular Ca2+ (P < 0.05); n = 8.

Effect of IP3 receptor inhibition in absence of extracellular Ca2+. Our next goal was to test the hypothesis that IP3-mediated release of Ca2+ from intracellular stores is involved in the PE-induced contractile response. We pretreated the strips with the IP3 receptor blocker 2-APB before PE stimulation in the absence of extracellular Ca2+ (Fig. 4). Inhibition of IP3 receptors with 2-APB (100 µM) abolished the early increase in [Ca2+]i in response to PE and reduced the early increase in tension by 43 ± 5% compared with that observed in the absence of extracellular Ca2+. 2-APB had no effect on the late PE-induced increase in tension.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 4.   A: Changes in [Ca2+]i and tension induced by 10 µM PE in the presence or absence (Ca2+ free) of 2-aminoethoxydiphenyl borate (2-APB; 100 µM) in a PASM strip. Extracellular Ca2+ concentration was 0 mM. B: summarized data. *Significantly different from response measured in the absence of extracellular Ca2+ (P < 0.05); n = 7.

Effect of PKC inhibition. To test the hypothesis that PKC activation plays a role in PE-induced increases in [Ca2+]i and tension, PASM strips were pretreated with the PKC inhibitor Bis I before PE stimulation. In the presence of extracellular Ca2+, pretreatment with Bis I (3 µM) had no effect on the early increase in [Ca2+]i but reduced the late increase in [Ca2+]i by 60 ± 3% (Fig. 5). Bis I reduced both the early (20 ± 5%) and late (33 ± 4%) increases in tension in response to PE. The inactive analog of Bis I (Bis V) had no effect on PE-induced increases in [Ca2+]i or tension. To further confirm a role for PKC in PE-induced contraction, concentration-response curves for KCl and PE were obtained in PASM rings in the presence or absence of another specific PKC inhibitor, calphostin C. Because of the light-sensitive nature of calphostin C, [Ca2+]i could not be measured in these studies. Pretreatment with calphostin C (1 µM) for 60 min had no effect on resting tension or KCl-induced contraction (Fig. 6). However, PE-induced contraction was inhibited by calphostin C, resulting in a 43 ± 5% decrease in maximal tension.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 5.   A: changes in [Ca2+]i and tension induced by 10 µM PE in the presence or absence (Control) of bisindolylmaleimide (Bis) I (3 µM) in a PASM strip. Extracellular Ca2+ concentration was 2.5 mM. B: summarized data. *Significantly different from response measured before PKC inhibition (P < 0.05); n = 6.



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 6.   Summarized data depicting the effect of calphostin C (Cal; 0.1, 0.3, 1.0 µM) on KCl (A) and PE (B) dose-response relationship in pulmonary arterial rings. Extracellular Ca2+ concentration was 2.5 mM. *Significantly different from response measured in the absence of calphostin C (P < 0.05); n = 10 in each group.

Effect of PKC inhibition in absence of extracellular Ca2+. We next tested the hypothesis that PKC activation is involved in regulating the PE-induced increase in tension observed in the absence of extracellular Ca2+. We pretreated strips with Bis I in the absence of extracellular Ca2+ before stimulation with PE. In the absence of extracellular Ca2+, Bis I (3 µM) had no effect on the early increase in [Ca2+]i induced by PE but reduced the early increase in tension by 25 ± 6% (Fig. 7). Bis I had no significant effect on the late PE-induced increase in tension.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 7.   A: changes in [Ca2+]i and tension induced by 10 µM PE in the presence or absence (Ca2+ free) of Bis I (3 µM) in a PASM strip. Extracellular Ca2+ concentration was 0 mM. B: summarized data. *Significantly different from response measured before PKC inhibition in the absence of extracellular Ca2+ (P < 0.05); n = 7.

Effect of PKC inhibition in absence of extracellular Ca2+ and IP3-mediated SR Ca2+ release. We tested the hypothesis that the increase in tension observed in the absence of any increase in [Ca2+]i is mediated by activation of PKC. PASM strips were pretreated with Bis I in the absence of extracellular Ca2+ and in the presence of 2-APB before stimulation with PE. Under these conditions, PE-induced increases in tension were not associated with concomitant increases in [Ca2+]i. As summarized in Fig. 8A, Bis I reduced both the PE-induced early (20 ± 5%) and late (18 ± 6%) increases in tension.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 8.   Changes in tension induced by 10 µM PE in the presence or absence of Bis I (A), tyrphostin (Tyr) A47 (B), combined Bis I and Tyr A47 (C), or Y-27632 (D) in PASM strips pretreated with 2-APB (100 µM). Extracellular Ca2+ concentration was 0 mM. *Significantly different from response measured before administration of the various inhibitors in the absence of extracellular Ca2+ and after inositol 1,4,5-trisphosphate receptor inhibition (P < 0.05); n = 5 in each group.

Effect of TK inhibition. To test the hypothesis that activation of TK plays a role in PE-induced increases in [Ca2+]i and tension, we pretreated PASM strips with the TK inhibitor Tyr A47 before PE stimulation. Pretreatment with Tyr A47 (10 µM) had no effect on the early increase in [Ca2+]i but, surprisingly, enhanced the late increase in [Ca2+]i by 79 ± 20% (Fig. 9). Tyr A47 had no effect on the early increase in tension but enhanced the late increase in tension by 20 ± 8%. The inactive analog of Tyr A47 (Tyr A1; 10 µM) did not alter PE-induced increases in [Ca2+]i or tension. In contrast to Tyr A47, pretreatment with genistein (100 µM) reduced the early increase in [Ca2+]i and tension by 55 ± 6% and 38 ± 12%, respectively. Genistein also reduced the late increases in [Ca2+]i and tension by 60 ± 8% and 20 ± 8%, respectively. The inactive analog of genistein (daidzein; 100 µM) had no effect on PE-induced increases in [Ca2+]i or tension.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 9.   A: changes in [Ca2+]i and tension induced by 10 µM PE in the presence or absence (Control) of Tyr A47 (10 µM) in a PASM strip. Extracellular Ca2+ concentration was 2.5 mM. B: summarized data. *Significantly different from response measured before tyrosine kinase (TK) inhibition (P < 0.05); n = 6.

Effect of TK inhibition in absence of extracellular Ca2+. We tested the hypothesis that activation of TK is involved in regulating the PE-induced increase in tension observed in the absence of extracellular Ca2+. PASM strips were pretreated with Tyr A47 in the absence of extracellular Ca2+ before stimulation with PE. Similar to Bis I, Tyr A47 (10 µM) had no effect on the early increase in [Ca2+]i in response to PE but reduced the early increase in tension by 22 ± 6% (Fig. 10). Tyr A47 had no effect on late changes in tension. Pretreatment with genistein (100 µM) reduced the early PE-induced increase in [Ca2+]i and tension by 68 ± 12% and 60 ± 16%, respectively. Genistein reduced the late phase of PE-induced contraction by 55 ± 15%.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 10.   A: changes in [Ca2+]i and tension induced by 10 µM PE in the presence or absence (Ca2+ free) of Tyr A47 (10 µM) in a PASM strip. Extracellular Ca2+ concentration was 0 mM. B: summarized data. *Significantly different from response measured before TK inhibition in the absence of extracellular Ca2+ (P < 0.05); n = 6.

Effect of TK inhibition in absence of extracellular Ca2+ and IP3-mediated SR Ca2+ release. To test the hypothesis that the increase in tension observed in the absence of any increase in [Ca2+]i is mediated by activation of TK, we pretreated strips with Tyr A47 in the absence of extracellular Ca2+ and in the presence of 2-APB before stimulation with PE. As noted above, under these conditions PE-induced increases in tension were not associated with concomitant increases in [Ca2+]i. As summarized in Fig. 8B, Tyr A47 reduced the early (34 ± 3%) and late (30 ± 5%) increases in tension in response to PE.

Effect of combined TK and PKC inhibition in absence of extracellular Ca2+ and IP3-mediated SR Ca2+ release. We also tested the hypothesis that the effects of PKC and TK on myofilament Ca2+ sensitization were additive (i.e., the signaling pathways are in parallel). PASM strips were pretreated with both Bis I and Tyr A47 in the absence of extracellular Ca2+ and in the presence of 2-APB before stimulation with PE. As summarized in Fig. 8C, the effect of combined PKC and TK inhibition on PE-induced contraction was no greater than that observed with Bis I or Tyr A47 alone.

Effect of ROK inhibition. To test the hypothesis that ROK plays a role in PE-induced increases in [Ca2+]i and tension, PASM strips were pretreated with the ROK inhibitor Y-27632 before PE stimulation. In the presence of extracellular Ca2+, pretreatment with Y-27632 (10 µM) reduced the early (21 ± 7%) but not the late increase in [Ca2+]i in response to PE (Fig. 11). However, pretreatment with Y-27632 reduced both the early and late PE-induced increases in tension by 52 ± 7% and 54 ± 8%, respectively.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 11.   A: changes in [Ca2+]i and tension induced by 10 µM PE in the presence or absence (Control) of Y-27632 (10 µM) in a PASM strip. Extracellular Ca2+ concentration was 2.5 mM. B: summarized data. *Significantly different from response measured before Rho kinase inhibition (P < 0.05); n = 5.

Effect of ROK inhibition in absence of extracellular Ca2+ and IP3-mediated SR Ca2+ release. Finally, to test the hypothesis that the increase in tension observed in the absence of any increase in [Ca2+]i is mediated by activation of ROK, we pretreated strips with Y-27632 in the absence of extracellular Ca2+ and in the presence of 2-APB before stimulation with PE. Under these conditions, inhibition of ROK with Y-27632 (10 µM) abolished PE-induced contraction (Fig. 8D).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To our knowledge, this is the first study to simultaneously measure PE-induced changes in [Ca2+]i and tension in PASM in the presence and absence of extracellular Ca2+ before and after inhibition of PKC, TK, ROK, and IP3 receptors. Our results indicate the following. Transsarcolemmal Ca2+ influx and myofilament Ca2+ sensitization contribute to both the early and late increases in tension. IP3 receptor-mediated release of Ca2+ from the sarcoplasmic reticulum contributes to the increase in early tension but not the late phase of tension. PKC and TK activation positively regulate both PE-induced Ca2+ influx and myofilament Ca2+ sensitization. The effects of PKC and TK on myofilament Ca2+ sensitivity were comparatively minor and not additive. Finally, ROK is the predominant pathway mediating PE-induced myofilament Ca2+ sensitization. However, it should be noted that the relative roles of these pathways may vary depending on the stimulus intensity and/or degree of resting tension. In addition, cross talk (30, 39) likely occurs between these pathways and may account for some of the differences observed in other studies (18, 42).

[Ca2+]i-tension relationship. Our first goal was to determine the relationship between [Ca2+]i and tension in response to membrane depolarization with KCl (electromechanical coupling), in which no phospholipid-derived second messengers are liberated, compared with that observed with alpha -adrenoreceptor stimulation with PE (pharmacomechanical coupling). KCl essentially caused monotonic increases in [Ca2+]i and tension. In contrast, PE caused a transient early increase in [Ca2+]i and tension, followed by a late phase of tension and [Ca2+]i that remained elevated above baseline but below peak values. Because the PE-induced increase in tension was similar to that of KCl, whereas the increase in [Ca2+]i was only about half of that observed with KCl, our results suggest that PE increases myofilament Ca2+ sensitivity. A PE-induced increase in myofilament Ca2+ sensitivity is also suggested by the continued contraction during the washout period when [Ca2+]i had returned to baseline and by the leftward shift in the continuous [Ca2+]i-tension relationship compared with KCl. A PE-induced leftward shift in the [Ca2+]i-tension relationship was also observed in rabbit PASM (12). We then investigated the roles of transsarcolemmal Ca2+ influx and IP3-mediated Ca2+ release in mediating the PE-induced increases in [Ca2+]i and tension.

Ca2+ influx and Ca2+ release. Removal of extracellular Ca2+ abolished the late increase in [Ca2+]i in response to PE, indicating that it is entirely dependent on transsarcolemmal Ca2+ influx. The early increase in [Ca2+]i was also modestly reduced, likely reflecting a small depletion in the Ca2+ content of the sarcoplasmic reticulum in response to removal of extracellular Ca2+. Both the early and late increases in tension were reduced when extracellular Ca2+ was removed, indicating that transsarcolemmal Ca2+ influx plays a major role in PE-induced contraction in PASM. For reasons that remain unclear, our results appear to be in direct contrast to a recent study that reported that removal of extracellular Ca2+ or administration of voltage-gated Ca2+ channel blockers had no effect on tension development in response to alpha -adrenoreceptor stimulation in endothelium-intact PASM (18). However, in a separate study, PE-induced contractions were found to be reduced by >75% in a nominally Ca2+-free buffer and involved a nisoldipine-insensitive, SK&F-96365-sensitive Ca2+ influx pathway (17). In that same study, depletion of IP3-sensitive Ca2+ stores, but not caffeine-sensitive Ca2+ stores, nearly abolished the PE response. This study by Jabr and coworkers (17) suggests that a nisoldipine-insensitive Ca2+ entry pathway is normally involved in filling IP3-sensitive Ca2+ stores and that IP3-sensitive and caffeine-sensitive Ca2+ stores are functionally separate and independent entities in PASM. In our study, Ca2+ influx could be mediated via activation of receptor-operated Ca2+ channels (9), by activation of voltage-gated Ca2+ channels in response to PE-induced membrane depolarization (35), or by capacitative Ca2+ entry in response to depletion of Ca2+ stores in the sarcoplasmic reticulum (8). In the absence of extracellular Ca2+, IP3 receptor inhibition abolished the early increase in [Ca2+]i, thereby indicating that Ca2+ release from IP3-sensitive Ca2+ stores in the sarcoplasmic reticulum was the mechanism responsible for the early increase in [Ca2+]i in response to PE. Our data are consistent with the findings of Jabr et al. (17) that both sarcolemmal Ca2+ influx and Ca2+ release from intracellular stores (IP3 sensitive) are involved in PE-induced contraction.

Myofilament Ca2+ sensitization. After removal of extracellular Ca2+ and inhibition of IP3 receptor-mediated Ca2+ release, PE increased tension without a concomitant increase in [Ca2+]i. The most likely explanation for this result is a PE-induced increase in myofilament Ca2+ sensitivity. An alternative explanation could be that there is a Ca2+-independent mechanism involved, rather than an increase in myofilament Ca2+ sensitivity. However, these two possibilities are not mutually exclusive and may exist in parallel or be linked to each other (i.e., Ca2+-independent pathway triggering an increase in myofilament Ca2+ sensitivity). This could be achieved through activation of PKC, TK, ROK, or a yet unidentified pathway. Thus we next investigated the roles of PKC, TK, and ROK in mediating this effect, as well as PE-induced changes in Ca2+ influx.

PKC. PKC activation is concomitant with IP3 production in response to agonist stimulation of phospholipase C (50). Alternatively, agonist activation of phospholipase D (1, 34) or phospholipase A2 (10, 38) can result in PKC activation in the absence of IP3 production. PKC has been reported to play a role in alpha -adrenoreceptor-mediated pulmonary vasoconstriction in isolated cat and rat lung (5, 22). In contrast, a recent report indicated that PKC is not involved in alpha -adrenoreceptor-mediated Ca2+ influx or contraction in endothelium-intact canine pulmonary artery (18). To resolve this controversy, we assessed the extent to which PKC mediates increases in [Ca2+]i and tension simultaneously in response to PE. PKC inhibition with Bis I attenuated the PE-induced late increases in [Ca2+]i and tension, which suggests that a component of the late increase in tension is mediated by PKC-dependent sarcolemmal Ca2+ influx. Bis I had no effect on KCl-induced increases in [Ca2+]i or tension, indicating that its effect was not due to some nonspecific action on myosin light chain kinase. Moreover, calphostin C, a structurally different PKC inhibitor, also attenuated PE-induced, but not KCl-induced, contraction. These data are consistent with other studies that demonstrated that PKC activation by phorbol esters enhances L-type channel-mediated Ca2+ influx in some smooth muscle cells (28) as well as alpha -adrenoreceptor-stimulated Ca2+ influx through voltage-gated Ca2+ channels in rat portal vein and tail artery (25). Our results clearly indicate that PKC activation is involved in alpha -adrenoreceptor-mediated contraction of canine PASM.

After removal of extracellular Ca2+ and pretreatment with 2-APB, the PE-induced increase in tension, without a concomitant increase in [Ca2+]i, likely reflects an increase in myofilament Ca2+ sensitivity. Under these conditions, Bis I attenuated the PE-induced increase in tension, albeit only by 20% (Fig. 7A). These results indicate that PKC activation plays a relatively minor role in the Ca2+-sensitizing effect of PE. Potential mechanisms could include PKC-dependent activation of Na+/H+ exchange and intracellular alkalinization causing myofilament Ca2+ sensitization (23) or PKC-dependent inhibition of myosin light chain phosphatase via phosphorylation of CPI-17, an inhibitor of the catalytic subunit on the phosphatase (27, 46). Alternatively, PKC-dependent activation of the thin filament accessory proteins caldesmon or calponin may be involved (3, 13, 33). However, the fact that PE increased tension in the absence of an increase in [Ca2+]i and after PKC inhibition indicates that additional signaling pathways are involved.

TK. Because TK activation has also been implicated in the contractile response to alpha -adrenoreceptor activation in the pulmonary circulation (8, 18, 42), we assessed the extent to which inhibition of TK regulates [Ca2+]i and tension in response to PE. Much to our surprise, pretreatment with Tyr A47 appeared to enhance the late increase in [Ca2+]i and tension in response to PE when extracellular Ca2+ was present (Fig. 9). Tyr A47 was shown to exert no effect on arginine vasopressin-induced increases in Ca2+ in A7r5 aortic smooth muscle cells (21). In contrast, other studies demonstrated that inhibition of TK with various Tyr derivatives results in decreased Ca2+ entry in vascular smooth muscle cells (21, 43, 51, 52). It should be noted that repetitive exposure to PE alone was associated with slightly potentiated late increases in [Ca2+]i and tension (Fig. 2). This effect may explain, at least in part, the late PE-induced increases in [Ca2+]i and tension after Tyr A47. Alternatively, the effects of Tyr A47 could potentially be explained by the finding that some TK inhibitors, including Tyr A47, actually enhance tyrosine phosphorylation (6, 21). To further investigate this unexpected finding, we assessed the effects of a more widely used and structurally different TK inhibitor, genistein. In contrast to Tyr A47, genistein attenuated both the early and late increases in [Ca2+]i and tension in response to PE. This effect was observed in the presence and absence of extracellular Ca2+. These results suggest that a component of PE-induced contraction is due to Ca2+ release and transsarcolemmal Ca2+ influx. Our results with genistein are consistent with our previous finding (8) that TK inhibition caused a rightward shift in the PE-induced concentration-response relationship in pulmonary arterial rings and provide additional support for the idea that TK plays a role in regulating alpha -adrenoreceptor-mediated increases in [Ca2+]i and tension in PASM strips.

We also observed that TK inhibition in the absence of sarcolemmal Ca2+ influx and after IP3 receptor block attenuated PE-induced contraction (Fig. 8B). Under these conditions, the PE-induced increase in tension likely involves a TK-dependent increase in myofilament Ca2+ sensitivity. However, the importance of TK activation in the myofilament Ca2+-sensitizing effect of PE appears to be relatively minor (similar to PKC), again suggesting that another signaling pathway may play a greater role in PE-induced Ca2+ sensitization.

Combined TK and PKC inhibition. Controversy surrounding the relative roles of PKC and TK in mediating the overall Ca2+-sensitizing effect of PE in the pulmonary vasculature (5, 18) led us to assess the extent to which combined inhibition of PKC and TK attenuated PE-induced Ca2+ sensitization. The effect of combined inhibition of PKC and TK on the PE-induced increase in tension in the absence of extracellular Ca2+ and in the presence of 2-APB was not additive, suggesting that PKC and TK were acting via a final common pathway. Inhibition of myosin light chain phosphatase is a possible candidate (46).

ROK. Recent evidence in a variety of smooth muscle preparations (16, 36), including PASM (18, 46), 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. ROK phosphorylates the regulatory subunit of myosin light chain phosphatase and inhibits its catalytic activity, thus resulting in increased myosin light chain phosphorylation, Ca2+ sensitization, and increased tension (46). In our study, inhibition of ROK with Y-27632 attenuated KCl-induced tension by 20%, with no concomitant effect on [Ca2+]i, indicating that Y-27632 may have some nonspecific inhibitory effect on myosin light chain kinase activity. When extracellular Ca2+ was present and intracellular Ca2+ stores were preserved, inhibition of ROK resulted in a slight reduction in the PE-induced increase in early [Ca2+]i but no effect on late [Ca2+]i. However, both early and late increases in tension in response to PE were attenuated. These data suggest that ROK positively regulates IP3-dependent SR Ca2+ release while having no significant effect on Ca2+ influx (16). The reduction in both PE-induced early and late tension is likely explained by a modest reduction in Ca2+ availability but primarily by a decrease in the sensitizing component of the late contraction. ROK inhibition abolished the contractile response to PE in the absence of extracellular Ca2+ and after IP3 receptor block, indicating that ROK activation is the predominant pathway mediating PE-induced Ca2+ sensitization (Fig. 8D). Activation of ROK by PE may be due to the GTPase RhoA (46) or the release of arachidonic acid in response to PE (37, 46).

Relative roles of signaling pathways. As noted above, the inhibitory effects of PKC and TK inhibition (alone and in combination) on PE-induced contraction in the absence of changes in [Ca2+]i were relatively modest and not additive, whereas ROK inhibition abolished PE-induced contraction under these conditions. ROK inhibition also blocked about half of the PE-induced contraction when Ca2+ was available. These results suggest that PKC and TK play a relatively minor role in mediating PE-induced late tension and that they act via a final common pathway. TK-dependent activation of ROK (18, 41) or PKC-dependent activation of ROK (32, 44) may explain these findings. PKC activation may serve dual roles in the PE response by positively regulating the L-type Ca2+ current (47, 53) and enhancing myofilament Ca2+ sensitivity, possibly via interactions with CPI-17 (27). TK activation also appears to positively regulate sarcolemmal Ca2+ influx in PASM, which is consistent with studies in other vascular beds (24, 29). TK positively regulates myofilament Ca2+ sensitivity, perhaps via direct phosphorylation of tyrosine residues on myosin light chain phosphatase (30, 39, 41). RhoA activation of ROK could exert a direct effect on myosin light chain phosphatase activity to enhance myofilament Ca2+ sensitivity (46). Phosphorylation of calponin by ROK (20), PKC (33), or TK (31) is another potentially important pathway. Further studies will be required to fully elucidate these interactions.

In summary, our results suggest that PE activates multiple signaling pathways that mediate alpha -adrenoreceptor contraction in PASM via modulation of both [Ca2+]i and myofilament Ca2+ sensitization. PKC and TK activation positively regulate sarcolemmal Ca2+ influx and also play a relatively minor role in the regulation of myofilament Ca2+ sensitization via a final common pathway. ROK activation is the primary mechanism for regulating myofilament Ca2+ sensitization in response to PE in PASM.


    ACKNOWLEDGEMENTS

This study was supported by National Heart, Lung, and Blood Institute Grants HL-38291 and HL-40361.


    FOOTNOTES

Address for reprint requests and other correspondence: P. A. Murray, Center for Anesthesiology Research, FF-40, 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.

July 12, 2002;10.1152/ajplung.00345.2001

Received 27 August 2001; accepted in final form 8 July 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Aburto, T, Jinsi A, and Deth RC. Involvement of protein kinase C activation in alpha 2-adrenoreceptor-mediated contractions of rabbit saphenous vein. Eur J Pharmacol 277: 35-44, 1995[ISI][Medline].

2.   Ascher-Landsberg, J, Saunders T, Elovitz M, and Phillippe M. The effects of 2-aminoethoxydiphenyl borate, a novel inositol 1,4,5-trisphosphate receptor modulator on myometrial contractions. Biochem Biophys Res Commun 264: 979-982, 1999[ISI][Medline].

3.   Carmichael, JD, Winder SJ, Walsh MP, and Kargacin GJ. Calponin and smooth muscle regulation. Can J Physiol Pharmacol 72: 1415-1419, 1994[ISI][Medline].

4.   Damron, DS, Nadim HS, Hong SJ, Darvish A, and Murray PA. Intracellular translocation of protein kinase C isoforms in canine pulmonary artery smooth muscle cells by angiotensin II. Am J Physiol Lung Cell Mol Physiol 274: L278-L288, 1998[Abstract/Free Full Text].

5.   De Witt, BJ, Kaye AD, Ibrahim IN, Bivalacqua TJ, D'Souza FM, Banister RE, Arif AS, and Nossaman BD. Effects of PKC isozyme inhibitors on constrictor responses in the feline pulmonary vascular bed. Am J Physiol Lung Cell Mol Physiol 280: L50-L57, 2001[Abstract/Free Full Text].

6.   Di Salvo, J, Nelson SR, and Kaplan N. Protein tyrosine phosphorylation in smooth muscle: a potential coupling mechanism between receptor activation and intracellular calcium. Proc Soc Exp Biol Med 214: 285-301, 1997[Abstract].

7.   DiSalvo, J, Steusloff A, Semenchuk L, Satoh S, Kolquist K, and Pfitzer G. Tyrosine kinase inhibitors suppress agonist-induced contraction in smooth muscle. Biochem Biophys Res Commun 190: 968-974, 1993[ISI][Medline].

8.   Doi, S, Damron DS, Horibe M, and Murray PA. Capacitative Ca2+ entry and tyrosine kinase activation in canine pulmonary arterial smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 278: L118-L130, 2000[Abstract/Free Full Text].

9.   Fasolato, C, Innocenti B, and Pozzan T. Receptor-activated Ca2+ influx: how many mechanisms for how many channels? Trends Pharmacol Sci 15: 77-83, 1994[ISI][Medline].

10.   Gailly, P, Gong MC, Somlyo AV, and Somlyo AP. Possible role of atypical protein kinase C activated by arachidonic acid in Ca2+ sensitization of rabbit smooth muscle. J Physiol 500: 95-109, 1997[Abstract].

11.   Haller, H, Smallwood JI, and Rasmussen H. Protein kinase C translocation in intact vascular smooth muscle strips. Biochem J 270: 375-381, 1990[ISI][Medline].

12.   Himpens, B, Kitazawa T, and Somlyo AP. Agonist-dependent modulation of Ca2+ sensitivity in rabbit pulmonary artery smooth muscle. Pflügers Arch 417: 21-28, 1990[ISI][Medline].

13.   Horowitz, A, Clement-Chomienne O, Walsh MP, and Morgan KG. Epsilon-isoenzyme of protein kinase C induces a Ca2+-independent contraction in vascular smooth muscle. Am J Physiol Cell Physiol 271: C589-C594, 1996[Abstract/Free Full Text].

14.   Horowitz, A, Menice CB, LaPorte R, and Morgan KG. Mechanisms of smooth muscle contraction. Physiol Rev 76: 967-1003, 1996[Abstract/Free Full Text].

15.   Ishizaki, T, Uehata M, Tamechika I, Keel J, Nonomura K, Maekawa M, and Narumiya S. Pharmacological properties of Y-27632, a specific inhibitor of rho-associated kinases. Mol Pharmacol 57: 976-983, 2000[Abstract/Free Full Text].

16.   Ito, S, Kume H, Honjo H, Kodama I, Yamaki K, and Hayashi H. Possible involvement of Rho kinase in Ca2+ sensitization and mobilization by MCh in tracheal smooth muscle. Am J Physiol Lung Cell Mol Physiol 280: L1218-L1224, 2001[Abstract/Free Full Text].

17.   Jabr, RI, Toland H, Gelband CH, Wang XX, and Hume JR. Prominent role of intracellular Ca2+ release in hypoxic vasoconstriction of canine pulmonary artery. Br J Pharmacol 122: 21-30, 1997[Abstract].

18.   Janssen, LJ, Lu-Chao H, and Netherton S. Excitation-contraction coupling in pulmonary vascular smooth muscle involves tyrosine kinase and Rho kinase. Am J Physiol Lung Cell Mol Physiol 280: L666-L674, 2001[Abstract/Free Full Text].

19.   Jin, N, Siddiqui RA, English D, and Rhoades RA. Communication between tyrosine kinase pathway and myosin light chain kinase pathway in smooth muscle. Am J Physiol Heart Circ Physiol 271: H1348-H1355, 1996[Abstract/Free Full Text].

20.   Kaneko, T, Amano M, Maeda A, Goto H, Takahashi K, Ito M, and Kaibuchi K. Identification of calponin as a novel substrate of Rho-kinase. Biochem Biophys Res Commun 273: 110-116, 2000[ISI][Medline].

21.   Kaplan, N, and Di Salvo J. Coupling between [arginine8]-vasopressin-activated increases in protein tyrosine phosphorylation and cellular calcium in A7r5 aortic smooth muscle cells. Arch Biochem Biophys 326: 271-280, 1996[ISI][Medline].

22.   Kaye, AD, Nossaman BD, Ibrahim IN, Feng CJ, and Kadowitz PJ. Influence of protein kinase C inhibitors on vasoconstrictor responses in the pulmonary vascular bed of cat and rat. Am J Physiol Lung Cell Mol Physiol 268: L532-L538, 1995[Abstract/Free Full Text].

23.   Krampetz, IK, and Rhoades RA. Intracellular pH: effect on pulmonary arterial smooth muscle. Am J Physiol Lung Cell Mol Physiol 260: L516-L521, 1991[Abstract/Free Full Text].

24.   Kusaka, M, and Sperelakis N. Inhibition of L-type calcium current by genistein, a tyrosine kinase inhibitor, in pregnant rat myometrial cells. Biochim Biophys Acta 1240: 196-200, 1995[ISI][Medline].

25.   Leprêtre, N, Mironneau J, and Morel JL. Both alpha 1A- and alpha 2A-adrenoreceptor subtypes stimulate voltage-operated L-type calcium channels in rat portal vein myocytes. J Biol Chem 269: 29546-29552, 1994[Abstract/Free Full Text].

26.   Levitzki, A. Tyrphostins---potential antiproliferative agents and novel molecular tools. Biochem Pharmacol 40: 913-918, 1990[ISI][Medline].

27.   Li, L, Eto M, Lee MR, Morita F, Yazawa M, and Kitazawa T. Possible involvement of the novel CPI-17 protein in protein kinase C signal transduction of rabbit arterial smooth muscle. J Physiol 508: 871-881, 1998[Abstract/Free Full Text].

28.   Litten, RZ, Suba EA, and Roth BL. Effects of a phorbol ester on rat aortic contraction and calcium influx in the presence and absence of BAY K 8644. Eur J Pharmacol 144: 185-191, 1987[ISI][Medline].

29.   Liu, H, and Sperelakis N. Tyrosine kinases modulate the activity of single L-type calcium channels in vascular smooth muscle cells from rat portal vein. Can J Physiol Pharmacol 75: 1063-1068, 1997[ISI][Medline].

30.   Martinez, MC, Randriamboavonjy V, Ohlmann P, Komas N, Duarte J, Schneider F, Stoclet JC, and Andriantsitohaina R. Involvement of protein kinase C, tyrosine kinases, and Rho kinase in Ca2+ handling of human small arteries. Am J Physiol Heart Circ Physiol 279: H1228-H1238, 2000[Abstract/Free Full Text].

31.   Masuda, H, Tanaka K, Takagi M, Ohgami K, Sakamaki T, Shibata N, and Takahashi K. Molecular cloning and characterization of human non-smooth muscle calponin. J Biochem (Tokyo) 120: 415-424, 1996[Abstract].

32.   Meacci, E, Donati C, Cencetti F, Romiti E, and Bruni P. Permissive role of protein kinase C alpha but not protein kinase C delta in sphingosine 1-phosphate-induced Rho A activation in C2C12 myoblasts. FEBS Lett 482: 97-101, 2000[ISI][Medline].

33.   Menice, CB, Hulvershorn J, Adam LP, Wang CA, and Morgan KG. Calponin and mitogen-activated protein kinase signaling in differentiated vascular smooth muscle. J Biol Chem 272: 25157-25161, 1997[Abstract/Free Full Text].

34.   Muthalif, MM, Parmentier JH, Benter IF, Karzoun N, Ahmed A, Khandekar Z, Adl MZ, Bourgoin S, and Malik KU. Ras/mitogen-activated protein kinase mediates norepinephrine-induced phospholipase D activation in rabbit aortic smooth muscle cells by a phosphorylation-dependent mechanism. J Pharmacol Exp Ther 293: 268-274, 2000[Abstract/Free Full Text].

35.   Nelson, MT, Patlak JB, Worley JF, and Standen NB. Calcium channels, potassium channels, and voltage dependence of arterial smooth muscle tone. Am J Physiol Cell Physiol 259: C3-C18, 1990[Abstract/Free Full Text].

36.   Nobe, K, and Paul RJ. Distinct pathways of Ca2+ sensitization in porcine coronary artery: effects of Rho-related kinase and protein kinase C inhibition on force and intracellular Ca2+. Circ Res 88: 1283-1290, 2001[Abstract/Free Full Text].

37.   Ogawa, K, Tanaka S, and Murray PA. Propofol potentiates phenylephrine-induced contraction via cyclooxygenase inhibition in pulmonary artery smooth muscle. Anesthesiology 94: 833-839, 2001[ISI][Medline].

38.   Parsons, SJ, Sumner MJ, and Garland CJ. Phospholipase A2 and protein kinase C contribute to myofilament sensitization to 5-HT in the rabbit mesenteric artery. J Physiol 491: 447-453, 1996[Abstract].

39.   Pfitzer, G, and Arner A. Involvement of small GTPases in the regulation of smooth muscle contraction. Acta Physiol Scand 164: 449-456, 1998[ISI][Medline].

40.   Rembold, CM, and Murphy RA. [Ca2+]-dependent myosin phosphorylation in phorbol diester stimulated smooth muscle contraction. Am J Physiol Cell Physiol 255: C719-C723, 1988[Abstract/Free Full Text].

41.   Sakurada, S, Okamoto H, Takuwa N, Sugimoto N, and Takuwa Y. Rho activation in excitatory agonist-stimulated vascular smooth muscle. Am J Physiol Cell Physiol 281: C571-C578, 2001[Abstract/Free Full Text].

42.   Savineau, JP, Gonzalez De La Fuente P, and Marthan R. Effect of modulators of tyrosine kinase activity on agonist-induced contraction in the rat pulmonary vascular smooth muscle. Pulm Pharmacol 9: 189-195, 1996[ISI][Medline].

43.   Semenchuk, LA, and Di Salvo J. Receptor-activated increases in intracellular calcium and protein tyrosine phosphorylation in vascular smooth muscle cells. FEBS Lett 370: 127-130, 1995[ISI][Medline].

44.   Slater, SJ, Seiz JL, Stagliano BA, and Stubbs CD. Interaction of protein kinase C isozymes with Rho GTPases. Biochemistry 40: 4437-4445, 2001[ISI][Medline].

45.   Somlyo, AP, and Somlyo AV. Signal transduction and regulation in smooth muscle. Nature 372: 231-236, 1994[ISI][Medline].

46.   Somlyo, AP, and Somlyo AV. Signal transduction by G-proteins, Rho-kinase and protein phosphatase to smooth muscle and non-muscle myosin II. J Physiol 522: 177-185, 2001[Abstract/Free Full Text].

47.   Suenaga, H, and Kamata K. Alpha-adrenoceptor agonists produce Ca2+ oscillations in isolated rat aorta: role of protein kinase C. J Smooth Muscle Res 36: 205-218, 2000[Medline].

48.   Toullec, D, Pianetti P, Coste H, Bellevergue P, Grand-Perret T, Ajakane M, Baudet V, Boissin P, Boursier E, and Loriolle F. The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. J Biol Chem 266: 15771-15781, 1991[Abstract/Free Full Text].

49.   Van Breeman, C, and Saida K. Cellular mechanisms regulating [Ca2+]i smooth muscle. Annu Rev Physiol 51: 315-329, 1989[ISI][Medline].

50.   Walsh, MP, Andrea JE, Allen BG, Clement-Chomienne O, Collins EM, and Morgan KG. Smooth muscle protein kinase C. Can J Physiol Pharmacol 72: 1392-1399, 1994[ISI][Medline].

51.   Wijetunge, S, Aalkjaer C, Schachter M, and Hughes AD. Tyrosine kinase inhibitors block calcium channel currents in vascular smooth muscle cells. Biochem Biophys Res Commun 189: 1620-1623, 1992[ISI][Medline].

52.   Wijetunge, S, and Hughes AD. pp60c-src increases voltage-operated calcium channel currents in vascular smooth muscle cells. Biochem Biophys Res Commun 217: 1039-1044, 1995[ISI][Medline].

53.   Xuan, YT, Wang OL, and Whorton A. Regulation of endothelin-induced Ca2+ mobilization in smooth muscle cells by protein kinase C. Am J Physiol Cell Physiol 266: C1560-C1567, 1994[Abstract/Free Full Text].


Am J Physiol Lung Cell Mol Physiol 283(5):L1051-L1064
1040-0605/02 $5.00 Copyright © 2002 the American Physiological Society