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Reconstitution of protein kinase C-induced contractile Ca²⁺ sensitization in Triton X-100-demembranated rabbit arterial smooth muscle

T. Kitazawa, N. Takizawa *, M. Ikebe * and M. Eto ¹

MS 9409 Received 22 March 1999; accepted after revision 24 June 1999.

	ABSTRACT

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1. Triton X-100-demembranated smooth muscle loses Ca²⁺-sensitizing responsiveness to protein kinase C (PKC) activators while intact and -toxin-permeabilized smooth muscles remain responsive. We attempted to reconstitute the contractile Ca²⁺ sensitization by PKC in the demembranated preparations.

2. Western blot analyses showed that the content of the PKC -isoform (PKC) was markedly reduced and that the smooth muscle-specific protein phosphatase-1 inhibitor protein CPI-17 was not detectable, while the amount of calponin and actin still remained similar to those of intact strips.

3. Unphosphorylated recombinant CPI-17 alone induced a small but significant contraction at constant Ca²⁺. Isoform-selective PKC inhibitors inhibited unphosphorylated but not pre-thiophosphorylated CPI-17-induced contraction, suggesting that in situ conventional PKC isoform(s) can phosphorylate CPI-17.

4. Exogenously replenishing PKC alone did not induce potentiation of contraction and only slowly increased myosin light chain (MLC) phosphorylation at submaximal Ca²⁺.

5. PKC in the presence of CPI-17, but not the [T38A]-CPI mutant, markedly induced potentiation of both contraction and MLC phosphorylation. CPI-17 itself was phosphorylated.

6. In in vitro experiments, CPI-17 was a much better substrate for PKC than calponin, caldesmon, MLC and myosin.

7. Our results indicate that PKC requires CPI-17 phosphorylation at Thr-38 but not calponin for reconstitution of the contractile Ca²⁺ sensitization in the demembranated arterial smooth muscle.

	INTRODUCTION

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The primary mechanism of smooth muscle contraction is reversible phosphorylation of myosin light chain (MLC) at Ser-19 (Kamm & Stull, 1985; Hartshorne, 1987). This phosphorylation was initially assumed to be a result of regulation of Ca²⁺/calmodulin-dependent MLC kinase by cytoplasmic Ca²⁺ only, since activity of phosphoprotein phosphatase toward MLC was presumed to be constant. It is now well established that physiological phosphorylation of MLC in smooth muscle is caused by not only an increase in the phosphorylating enzyme activity but also a decrease in the dephosphorylating enzyme activity through G protein(s) (Kitazawa et al. 1991b; Kubota et al. 1992).

Regulation of the MLC phosphatase is one of the current topics in smooth muscle research as well as an important field in general cell biology (Somlyo & Somlyo, 1994; Hartshorne et al. 1998). During stimulation of smooth muscle contraction, MLC phosphatase activity is reduced to increase the contractile Ca²⁺ sensitivity (Ca²⁺ sensitization) and this is possibly mediated by multiple second messengers/ signalling pathways: the rho A-rho kinase (Kimura et al. 1996), protein kinase C (PKC; Masuo et al. 1994) and arachidonic acid pathways (Gong et al. 1992). In contrast cyclic GMP, a vasodilator second messenger, has been demonstrated to increase MLC phosphatase activity and reduce the contractile Ca²⁺ sensitivity (Ca²⁺ desensitization) during vascular smooth muscle relaxation (Wu et al. 1996; Lee et al. 1997). During stimulation in smooth muscle, protein kinase and phosphatase targeting the same protein MLC work co-operatively to effect a response rather than working to achieve homeostasis by counterbalancing each other. Yet the precise mechanism of each signalling pathway regulating MLC phosphatase remains unclear.

The activation of PKC by diacylglycerol (DAG) analogues and phorbol esters in intact and permeabilized arterial smooth muscle instructs the MLC phosphatase to decrease its activity but has no effect on MLC kinase activity (Itoh et al. 1993; Masuo et al. 1994). The resultant gain in MLC phosphorylation at Ser-19 thus increases the Ca²⁺ sensitivity of smooth muscle contraction. The precise mechanism for the modulation of MLC phosphatase by PKC remains unknown, although in vitro evidence controverts the direct phosphorylation of MLC phosphatase by PKC (Hartshorne et al. 1998). CPI-17, a novel smooth muscle-specific regulatory protein, has been demonstrated to be phosphorylated by PKC, thereby inhibiting in vitro and in situ MLC phosphatase and increasing the Ca²⁺ sensitivity of both MLC phosphorylation and contraction (Eto et al. 1995, 1997; Li et al. 1998; Senba et al. 1999). This protein is postulated to be a mediator between PKC activation and inhibition of MLC phosphatase. There are several reports, however, showing that phorbol esters and agonists evoked a contraction of intact smooth muscle without an associated increase in MLC phosphorylation under Ca²⁺-free conditions (see review by Singer, 1996). It has been suggested that PKC activation may relieve the MLC phosphorylation-independent inhibition of contraction by affecting the thin filament-associated proteins caldesmon and/or calponin (see reviews by Allen & Walsh, 1994). These proteins can be phosphorylated in vitro by several protein kinases including PKC, but the evidence for in situ phosphorylation and its significance is conflicting (see review by Horowitz et al. 1996).

Several reports have shown that direct application of PKC enzyme in heavily permeabilized smooth muscles, which still retained the regulatory/contractile apparatus including MLC kinase and phosphatase, failed to cause an increase in contractile force levels at submaximal levels of Ca²⁺ (Inagaki et al. 1987; Sutton & Haeberle, 1990; Parente et al. 1992). This is in contrast to the Ca²⁺-sensitizing effect of PKC activators in intact and mildly (-toxin- or -escin-) permeabilized smooth muscles (Masuo et al. 1994). In this study, we first confirmed the previous results, that our Triton X-100-demembranated arterial strips lose contractile responsiveness to PKC activators, and then examined what constituents remain in the preparations using Western immunoblot analysis. Finally, we attempted to reconstitute the Ca²⁺-sensitizing response of the demembranated strips to active PKC.

	METHODS

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Tissue preparation and force measurement

All animal procedures were approved by the Animal Care and Use Committee of Georgetown University. Male New Zealand White rabbits (2·5-3·5 kg) were killed by inhalation of an overdose of halothane and exsanguinated. Smooth muscle strips (70 µm thick, 700-800 µm wide and 3 mm long) were dissected from rabbit femoral arteries and carefully freed of connective tissue, and their endothelia removed by rubbing with a razor blade as previously described (Kitazawa et al. 1991a). The strips were then tied with silk monofilaments to the fine tips of two tungsten needles, one of which was connected to a force transducer, and mounted over a well filled with solution on a Teflon-coated bubble plate to allow for moderately rapid (within a second) solution exchange and freezing. Experiments were carried out at 20°C except when mentioned elsewhere.

Solutions

The standard relaxing solution (Kitazawa et al. 1991a), used for resting states of the permeabilized strips, contained the following: 74·1 mM potassium methanesulfonate, 2 mM Mg²⁺, 4·5 mM MgATP, 1 mM EGTA, 10 mM creatine phosphate (CP), 30 mM Pipes, 1 mM dithiothreitol (DTT) and 0·1 % fatty acid-free BSA. At times we used slightly modified relaxing solutions, in which the concentration of EGTA was different. In the activating solution, 10 mM EGTA was used and a calculated amount of calcium methanesulfonate was added to give the final desired concentration of free Ca²⁺ ions (Kitazawa et al. 1991a). All solutions were neutralized to pH 7·1 with KOH at 20°C and an ionic strength of 0·2 M was achieved by appropriately using more or less potassium methanesulfonate.

Cell permeabilization

After measuring the force of contractions induced by high K ⁺ (154 mM) and by phenylephrine (100 µM) in freshly dissected strips, these strips were incubated in the standard relaxing solution for several minutes. For plasma membrane permeabilization with -toxin, the strips were treated for 30 min at 30°C with 20 µg ml^-1 of purified Staphylococcus aureus -toxin (List, Campbell, CA, USA) at pCa 6·7 buffered with 10 mM EGTA and then treated with 10 µM Ca²⁺ ionophore A23187 (Calbiochem) for 20 min at 25°C to deplete the SR of Ca²⁺ and keep the cytoplasmic Ca²⁺ constant (Masuo et al. 1994). For membrane permeabilization with -escin (Sigma), the strips were treated with 60 µM in standard relaxing solution for 45 min at 5°C and then for 20 min at 30°C in the presence of 10 µM A23187 (Masuo et al. 1994). To heavily permeabilize strips, we used 0·1 % Triton X-100 (Sigma) in standard relaxing solution for 30 min at 5°C and for 10 min at 30°C.

Western blot analyses

Intact or permeabilized femoral artery strips were fixed with ice-cold acetone containing 10 % trichloroacetic acid and kept at -20°C overnight. The strips were transferred to fresh acetone to remove trichloroacetic acid and dried. The dried strips were placed in boiled SDS sample buffer containing 62·5 mM Tris (pH 6·8), 1 % SDS, 15 % glycerol, 15 mM DTT and 0·01 % Bromophenol Blue, and ground with a glass-glass homogenizer. For CPI-17 Western blotting, a volume of the homogenate equivalent to two strips, for PKC blotting a volume equivalent to five strips, or for -smooth muscle actin and h-calponin a volume equivalent to 1/20th of one strip was run in a SDS-polyacrylamide slab gel. Proteins were transferred to nitrocellulose membranes. The membranes were rinsed with Tris-buffered saline (TBS) solution containing 0·05 % Tween 20 and blocked with 1 % gelatin in TBS-Tween soution for 1 h and then incubated with a primary antibody overnight with gentle rocking. Following washes with TBS-Tween solution, the membranes were incubated for 2 h at room temperature with a secondary antibody conjugated with alkaline phosphatase (Chemicon International, Temecula, CA, USA) at 1:5000 dilution in TBS-Tween solution containing 5 % non-fat milk. After final washes with TBS-Tween solution for 30 min, the membranes were developed with an alkaline phosphatase substrate solution containing bromochloroindolyl phosphate and nitro blue tetrazolium (Sigma) to visualize immunoreactive proteins. The dark purple band patterns of alkaline phosphatase product were digitized with a colour scanner (Macintosh) and analysed with image processing software (Signal Analytics Co., Vienna, VA, USA) that permitted the subtraction of background obtained from regions adjacent to the protein bands.

A polyclonal anti-CPI-17 antibody raised in rabbit, a monoclonal anti--smooth muscle actin (clone no. 1A4, Sigma) and a monoclonal anti-calponin (clone no. hCP, Sigma) were used at 1:5000 dilution. A monoclonal anti-PKC antibody was obtained from Transduction Laboratories and used at 1:500 dilution in the TBS solution.

Expression and purification of recombinant proteins

For the CPI-17, the recombinant hexahistidine-tagged protein was expressed in E. coli and purified to homogeneity as described previously (Eto et al. 1997). Thiophosphorylation of the CPI-17 was carried out as described previously (Li et al. 1998).

For PKC enzyme, two different mutants of rat PKC -isoform were expressed and purified (Takizawa et al. 1998). One PKC mutant lacked the entire C1 domain that corresponds to the codon 1-152. The second mutant lacked the N-terminal V1 region corresponding to the codon 1-18 and in addition, Arg-19 was replaced with Met. Briefly, the cDNA of each mutant was inserted into the baculovirus transfer vector PbluebacHis2 (Invitrogen, Carlsbad, CA, USA) containing a multi-histidine sequence. The recombinant viruses were produced as described previously (Matsu-ura & Ikebe, 1995). Sf9 cells were infected with the virus and harvested 3 days later. Mutants of PKC were extracted and purified with DE52 chromatography followed by NiNTA-agarose (Qiagen) chromatography. The proteins were eluted with 0·25 M imidazole buffer (pH 7·0). Each purified mutant of hexahistidine-tagged PKC showed a single band on SDS-PAGE gel. One unit of the activity is defined as the amount of enzyme that transfers 1 nmol of phosphate to histone H3 per minute at 25°C.

Protein phosphorylation

Phosphorylation levels of in situ 20 kDa MLC in permeabilized preparations were measured as described previously (Kitazawa et al. 1991a). Briefly, the strips were rapidly frozen with liquid N₂-cooled liquid chlorodifluoromethane, with force monitored up to the time of freezing. The various phosphorylated states of MLC, un- (U), mono- (P1) and di- (P2), were separated by two-dimensional isoelectric focusing/SDS-PAGE, blotted onto a nitrocellulose membrane, stained with colloidal gold to produce separate spots for each phosphorylated state, and the amount at each spot was measured by its density as described previously (Kitazawa et al. 1991a). The percentage of MLC phosphorylation was calculated by dividing (P1 + P2) by (U + P1 + P2).

For measurement of CPI-17 phosphorylation, the reaction was terminated by addition of same volume of 20 % trichroloacetic acid (TCA). After TCA was removed with acetone, dried precipitates were homogenized in a solution containing 8 M urea, 50 mM Tris-Cl (pH 8·0), 10 % 2-mercaptoethanol, 0·002 % Bromophenol Blue and 1 mM benzamidine. Phosphorylated CPI-17 was separated from the unphosphorylated form by electrophoresis using a gel containing 8 M urea, 100 mM Mops (pH 8·0 by NaOH) and 12 % acrylamide and 0·32 % N,N'-methylene bisacrylamide (Eto et al. 1997). The electrophoresis was performed from anode to cathode in the running buffer solution containing 50 mM Mops and 50 mM 2,6-lutidine under a constant voltage. Under these conditions, phosphorylated CPI-17 migrates more slowly than, and indeed separates from, unphosphorylated CPI-17. The extent of phosphorylation of CPI-17 was determined from the ratio of the band intensities of phosphorylated to total (unphosphorylated + phosphorylated) CPI-17 on the gel stained with Coomassie Brilliant Blue R250.

To measure the PKC phosphorylation activity toward various smooth muscle proteins, the reaction was carried out at 25°C in a solution containing 30 mM Tris-HCl (pH 7·5), 0·2 mM [-³²P]ATP, 30 mM KCl, 2 mM MgCl₂, 200 µM CaCl₂, 50 ng ml^-1 phorbol 12-myristate 13-acetate (PMA), 0·1 mg ml^-1 phosphatidylserine, 4·1 nM hexahistidine-tagged recombinant rat PKC wild-type and 5 µM substrate (gizzard calponin, caldesmon, MLC and myosin and recombinant porcine CPI-17). The reaction was terminated by transferring the assay mixture to a solution containing 5 % TCA and 1 % sodium pyrophosphate. The ³²P incorporation was measured as described (Walsh et al. 1983).

Chemicals

Phorbol-12,13-dibutyrate (PDBu) was from Gibco BRL; PMA, diC₆ and phosphatidylserine (PS) were from Sigma; calmodulin, PKC-IP and Gö6850 (GF109203X) were from Biomol (Plymouth Meeting, PA, USA); Gö6976 and human recombinant PKC were from Calbiochem; creatine phosphokinase, 4-(2-aminoethyl)-benzensulfonyl fluoride (Pefabloc), N-[N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]-agmatine (E-64), calpain inhibitor II from Boehringer Mannheim GmbH (Mannheim, Germany).

Statistics

All values are expressed as means ± S.E.M. Student's unpaired two-tailed t test was used for statistical analysis of the data and P < 0·05 was considered to be significant.

	RESULTS

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Effectiveness of PKC activators on contractile force at given levels of Ca²⁺ in -toxin, -escin- or Triton X-100-permeabilized smooth muscle

Figures 1 and 2 illustrate the different Ca²⁺-sensitizing efficacies of saturated concentrations of phorbol ester (3 µM phorbol-12,13-dibutyrate; PDBu) and synthetic short chain DAG (10 µM 1,2-sn-dihexanoyl-glycerol; diC₆), respectively, on three different permeabilized preparations of rabbit femoral artery smooth muscle strips. The first groupwas treated with Staphylococcus aureus -toxin and A23187 (Fig 1A and Fig 2A), the secondwith the saponin ester -escin and A23187 (Fig 1B and Fig 2B), and the thirdwith Triton X-100 alone (Fig 1C and Fig 2C) (see Methods for more details). All preparations, although their Ca²⁺ sensitivities were varied presumably due to different degrees of calmodulin loss, could reversibly produce a contraction in response to Ca²⁺ levels.

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Figure 1. Different efficacies of phorbol ester PDBu on contractile Ca2+ sensitivity in -toxin-, -escin- or Triton X-100-permeabilized arterial smooth muscle Strips of rabbit femoral artery were permeabilized with staphylococcal -toxin + A23187 (A), -escin + A23187 (B), or Triton X-100 (C) and then incubated in the relaxing solution (see Methods). They were placed in a submaximal pCa 6·7 (A), 6·3 (B) or 6 (C) solution until reaching a low steady-state level of force at 20 °C. Three micromolar PDBu was applied into the same pCa solution. After a steady-state level of force was reached, PDBu was washed out and a pCa 4·5 solution was added to verify maximum force levels in the preparations. D, summary of 4-6 independent experiments for each permeabilized preparation. The filled and hatched bars represent, respectively, relative force levels before and after addition of PDBu.

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Figure 2. Different efficacies of a synthetic short chain of DAG diC6 on contractile Ca2+ sensitivity in different permeabilized arterial smooth muscles Experiments were similar to those described in Fig. 1 except 10 µM diC6 was used as a PKC activator. The pCa of the activation solution was 6·7 in -toxin- (A), 6·3 in -escin- (B) or 6 in Triton X-100-treated preparations (C). D, summary of 3-6 independent experiments for each permeabilized preparation. The filled and hatched bars represent, respectively, relative force levels before and after addition of diC6.

Figures 1A and 2A demonstrate that, respectively, PDBu and diC₆ markedly potentiated a small contraction at constant Ca²⁺ (pCa 6·7) in -toxin-permeabilized preparations. These contractions were similar to that of intact strips before permeabilization (not shown), indicating that the large Ca²⁺ sensitization of contraction by PKC activators found in intact strips is not lost. In -escin-permeabilized preparations, both activators elicited a significant but much smaller potentiation of contraction at pCa 6·3 than that seen in -toxin-permeabilized strips (Fig 1B and Fig 2B). We have previously shown using the same smooth muscle tissue type permeabilized with either -toxin or -escin that contraction and MLC phosphorylation at Ser-19 were significantly increased by the same PKC activators at a given level of Ca²⁺ (Masuo et al. 1994). These activators, however, did not significantly evoke any contractile effect on Triton X-100-demembranated preparations (Fig 1C and Fig 2C).

An increase in concentration of PDBu to 30 µM did not alleviate the loss of the Ca²⁺-sensitizing response. Supplement of 0·5 µM (a concentration equivalent to EC₅₀) exogenous calmodulin markedly increased the Ca²⁺ sensitivity of contraction by 4-fold in Triton X-100-demembranated smooth muscle, but did not affect the maximal level of contraction at pCa 4·5. Adding calmodulin, however, did not restore the lost sensitizing effect of PDBu and diC₆ (not shown). The presence of exogenous creatine phosphokinase (0·5 mg ml^-1) affected neither contractile Ca²⁺ sensitivity nor the effect of PDBu and diC₆ on the Triton X-100-demembranated arterial smooth muscle contractions. Figures 1D and 2D show a statistical summary of contractile effects of PDBu and diC₆, respectively, on the different permeabilized preparations.

Western blots of actin, calponin, PKC and CPI-17

The contents of several proteins known to be important for smooth muscle contraction of rabbit femoral artery strips were examined using Western immunoblot analysis. After treatment with 0·1 % Triton X-100, the -smooth muscle actin in the arterial strips was slightly reduced to 80 ± 13 % (n = 4) of that found in non-permeabilized intact strips. The content of h-calponin in the demembranated arterial strips was found not to differ significantly from that of the intact strips (Fig. 3A); the density ratio of calponin to -smooth muscle actin was 0·59 ± 0·12 (n = 4) in intact and 0·72 ± 0·15 (n = 4) in Triton X-100-demembranated strips. The content of the conventional -isoform of PKC (PKC) was largely decreased by the detergent treatment to 10 ± 2 % (n = 3) of that in intact strips (Fig. 3B). Relative content of CPI-17 in -toxin-permeabilized strips did not significantly differ from that of intact strips. In contrast, -escin permeabilization significantly reduced the amount of CPI-17 to 35 ± 6 % (n = 4) of that in intact strips, while Triton X-100-demembranation caused a reduction in CPI-17 content to an undetectable level; no CPI-17 bands were detected in four independent demembranation experiments. A threefold increase in the amount of Triton X-100-treated sample applied did not cause the CPI-17 band to become visible. Figure 3C illustrates typical Western blots and Fig. 3D shows mean digitized results using anti-CPI-17 antibody in intact, -toxin-, -escin- and Triton X-100-permeabilized rabbit arterial smooth muscle strips.

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Figure 3. Contents of various smooth muscle-specific proteins in Triton X-100-demembranated arterial smooth muscle Western immunoblots were used to identify -smooth muscle actin (A), h-calponin (A; CaP), conventional PKC isoform (B; PKC) and CPI-17 (C) in Triton X-100-demembranated arterial smooth muscle strips as compared with those in untreated intact, or -toxin- or -escin-permeabilized preparations. A 15 % SDS-PAGE gel was used for actin, CaP and CPI-17, a 10 % gel for PKC. These images are representative of 3-4 similar experiments. D, a statistical summary of digitized results of four CPI-17 blot experiments in intact, -toxin-, -escin- and Triton X-100-permeabilized strips. (See Methods for more details.)

Unphosphorylated CPI-17-induced potentiation of contraction in Triton X-100 demembranated preparations

Five micromolar unphosphorylated CPI-17 (u-CPI) slightly but significantly (P < 0·01) enhanced a contraction induced by submaximal Ca²⁺ (pCa 6) from 11 ± 1 % (n = 16) to 20 ± 2 % (n = 7) of the maximal contraction at pCa 4·5 (Fig. 4A; also see Li et al. 1998). We believe this response to be a consequence of phosphorylation of u-CPI by the small amount of PKC remaining after demembranation rather than a direct effect of the unphosphorylated form of the protein. Pseudosubstrate inhibitory peptide of PKC (corresponding to amino acid sequence of 19-31; PKC-IP) at 30 µM fully suppressed this potentiation (Fig. 4B), but affected neither the basal contraction induced by Ca²⁺ alone nor a contraction (76 ± 4 % of the maximum, n = 4) stimulated by 5 µM pre-thiophosphorylated CPI-17 (p-CPI), which is not capable of being dephosphorylated in these cells. The u-CPI-induced contraction was also blocked by 10 µM Gö6976 (Martiny-Baron et al. 1993), a selective inhibitor for the conventional-PKC isoform (Fig. 4C), and also significantly inhibited by 1 µM Gö6850, a non isoform-selective PKC inhibitor (identical with GF109203X, not shown). [T38A]-CPI has a single mutation where threonine at position 38 is replaced with alanine. Unlike the wild-type, the unphosphorylated [T38A]-CPI mutant even at 20 µM did not cause an increase in force, suggesting together with the effect of PKC inhibitors that the contraction induced by unphosphorylated CPI-17 results from the phosphorylation of Thr-38 by PKC remaining in the demembranated preparations. In contrast, activation of any remaining PKC by 3 µM PDBu did not significantly further enhance the u-CPI-induced contraction (Fig. 4D). The presence of a protease inhibitor cocktail (1 mM benzamidine, 1 mM 4-(2-aminoethyl)-benzensulfonyl fluoride, 10 µM N-[N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]-agmatine and 25 µM calpain inhibitor II) throughout the experiments did not modify the development of u-CPI-induced contraction (not shown).

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Figure 4. Unphosphorylated CPI-induced contractions of Triton X-100-demembranated strips at a given level of Ca2+ A, potentiation of contraction by 5 µM u-CPI at pCa 6. The u-CPI-induced contraction was inhibited by the presence of selective PKC inhibitor 30 µM PKC-IP (B) or 10 µM Gö6976 (C). Three micromolar PDBu did not affect significantly the u-CPI-induced contraction (D). These traces are representative of 4 similar experiments.

Contractile effect of active PKC on Triton X-100-demembranated preparations

Three different forms of active PKC were used to replenish the lost PKC enzymes in Triton X-100-demembranated arterial strips. One, PKC-1, is a short (molecular mass = 64·7 kDa) mutant of hexahistidine-tagged -isoform, missing pseudosubstrate sequence and C1 domain of the N-terminal regulatory region and is therefore constitutively active in the absence of DAG. Phorbol 12-myristate 13-acetate (PMA) had no effect on the activity. This mutant phosphorylates its substrate, histone H3, at an activity of 256 U mg^-1 in the presence of 200 µM Ca²⁺. The activity of PKC-1 in the absence of phosphatidylserine (PS) is much greater than the wild-type, while the addition of PS further activates the enzyme. We used it at 0·8 µg ml^-1 in the absence of PS in the pCa 6 solution (Fig. 5A and C). The second PKC is a Ca²⁺- and DAG-dependent mutant of hexahistidine-tagged PKC, PKC-2, (molecular mass = 80·1 kDa) that lacks only a small portion in the N-terminal region. The activity of PKC-2 to histone H3 is 120 U mg^-1 in the presence of 0·1 mg ml^-1 PS and 200 µM Ca²⁺. We used this enzyme at 2·5 µg ml^-1 in the presence of 0·1 mg ml^-1 PS and 10 µM diC₆ in the pCa 6 solution (not shown), because PKC fragments containing only the catalytic domain may differ in substrate specificity from the enzyme with an intact regulatory domain (Nakabayashi et al. 1991). The third is a novel recombinant -isoform, PKC (800 U mg^-1 at 30°C as indicated in the manufacturer's specifications), whose activity is Ca²⁺ independent, but still requires PS and DAG; we used this at 2·5 µg ml^-1 in the presence of PS and diC₆ (Fig. 5D). None of the active PKC forms caused a significant increase in force levels at pCa 6 up to 20 min at 20°C (Fig. 5A and D). To confirm that PKCs were penetrating into myofibrils of Triton X-100-demembranated arterial strips, we applied a 150 kDa immumoglobulin that constitutively activates MLC kinase without an increase in Ca²⁺ and calmodulin (Araki & Ikebe, 1991). The anti-MLC kinase antibody (LKH18), which is substantially larger than any of the PKCs, did potentiate a contractile force at pCa 6, slowly but largely, from 12 ± 5 % (n = 4) to 62 ± 9 % (n = 4) of maximal Ca²⁺-activated contraction at pCa 4·5 (Fig. 5B), but had no contractile effect on the -toxin-permeabilized preparations (not shown). Thus, active PKC itself could not elicit contractile Ca²⁺ sensitization in myofibrils that have not lost their ability to be sensitized.

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Figure 5. Effects of active PKC and , anti-MLC kinase antibody and u-CPI on Triton X-100-demembranated smooth muscle contraction A 64·7 kDa mutant of PKC (PKC-1) alone at 0·8 µg ml-1 had no effect on a steady-state submaximal contraction at pCa 6 (A), indicating no Ca2+ sensitization. A monoclonal 150 kDa antibody against MLC kinase (LKH18) in vitro activates the kinase activity in the absence of Ca2+-calmodulin. The LKH18 at 11 µg ml-1 significantly (P < 0·01) enhanced the pCa 6 contraction (B), showing the Ca2+ sensitization. The same concentration of PKC-1 potentiated the contraction in the presence of 5 µM recombinant u-CPI (C), indicating PKC-induced sensitization. A novel -isoform of human recombinant PKC (PKC) alone at 2·5 µg ml-1 had no effect of the basal pCa 6-induced contraction (no sensitization), but addition of 5 µM u-CPI into the solution produced a contraction (sensitization) in Triton X-100-demembranated smooth muscle strips (D). These traces are representative of 4-7 similar experiments.

As mentioned above, u-CPI alone at 5 µM slightly but significantly increased force levels at constant Ca²⁺ (Fig 4A and Fig 5C). Addition of PKC-1 or PKC-2 with PS plus diC₆, each of which had no effect on the pCa 6 contraction, markedly potentiated the contractile force in the presence of u-CPI at the same Ca²⁺ level (Fig. 5C); PKC-1 increased force levels from 21 ± 1 (n = 5) to 64 ± 6 % (n = 4) and PKC-2 increased them from 18 ± 3 (n = 4) to 44 ± 6 % (n = 4) of maximal Ca²⁺-activated contraction. PKC and 5 µM u-CPI together led to 45 ± 5 % (n = 4) of maximal contraction (Fig. 5D). These potentiated levels were smaller than the force level achieved by the same concentration of thiophosphorylated p-CPI (see Fig. 4B and Li et al. 1998). Figure 6 illustrates a summary of the contractile effects of PKC-1.

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Figure 6. Summary of contractile effects of PKC-1 and u-CPI on Triton X-100-demembranated arterial smooth muscle and phosphorylation of CPI-17 by PKC (inset) The experiments were carried out by the method similar to that used in Fig. 5A and C. Relative force levels were measured as a percentage of the maximum contraction at pCa 4·5. *P < 0·01 compared with control; n = 4-7. Inset: Coomassie Brilliant Blue-stained urea-Mops gel. PKC-2 (2·5 µg ml-1) and u-CPI (5 µM) were applied for 0 and 5 min in the activating solution (pCa 6 + PS + diC6) surrounding Triton X-100-demembranated arterial strips mounted on a bubble plate. The reaction was terminated by transferring an aliquot of the mixture to a solution containing 20 % TCA. The urea-Mops gel electrophoresis was performed to measure phosphorylation levels of CPI-17 as described in the Methods. Only basic CPI-17 but not BSA or PKC in the solution was able to penetrate into the gel.

It has been shown previously that CPI-17 can be phosphorylated by PKC (Eto et al. 1995, 1997). In these strip experiments we found using the urea-Mops gel (see Methods) that most of the CPI-17 molecules were already phosphorylated 5 min after the same concentration of PKC-2 was added to the pCa 6 solution containing 5 µM u-CPI, PS and diC₆ at 20°C (see inset of Fig. 6). Although the primary sequence suggests multiple phosphorylation sites, threonine at position 38 is believed to be the phosphorylatable site critical to the function of inhibiting MLC phosphatase in vitro (Eto et al. 1997). [T38A]-CPI has a single point mutation where threonine at position 38 is replaced with alanine. Unlike the wild-type, the unphosphorylated [T38A]-CPI at 5 µM did not allow for large PKC-1- or PKC-2-induced potentiation of contraction at pCa 6 (Fig. 7A). Most of the mutant proteins were phosphorylated 20 min after addition of the PKC-2 (not shown), supporting the notion that Thr-38 is the single critical phosphorylation site.

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Figure 7. Effects of mutant CPI-17, denatured PKC-1 and selective PKC inhibitor peptide A, [T38A]-CPI is a mutant CPI-17 in which phosphorylatable threonine at position 38 is replaced with unphosphorylatable alanine. Neither 5 µM [T38A]-CPI alone nor with 0·8 µg ml-1 PKC-1 potentiated the pCa 6 contraction. B, denatured PKC-1 at 0·8 µg ml-1 in the presence of 5 µM u-CPI did not enhance a contraction at pCa 6. C, 30 µM PKC-IP inhibited contractions induced by 5 µM u-CPI alone and by u-CPI plus 0·8 µg ml-1 PKC-1. These traces are representative of 3-4 similar experiments.

Denatured PKC-1, produced by boiling for 5 min, prevented the increased force levels otherwise seen in the presence of 5 µM u-CPI (Fig. 7B). PKC-IP, the pseudosubstrate inhibitor peptide, at 30 µM, inhibited both the large potentiation of contraction at constant pCa 6 induced by PKC-1 plus u-CPI and the small potentiation induced by u-CPI alone (Fig. 7C).

Effect of active PKC on MLC phosphorylation in Triton X-100-demembranated preparations

MLC phosphorylation levels at pCa 6 alone for 15 min in the Triton X-100-demembranated strips were 10 ± 1 % (n = 7) of total MLC and, like the contractions, were not significantly increased by 0·8 µg ml^-1 PKC-1 (12 ± 2 %, n = 6) or 2·5 µg ml^-1 PKC-2 (13 ± 3 %, n = 4) after 5 min at 20°C (Fig. 8). However, contrary to what might be expected from the small stimulatory effect seen on contraction, 5 µM u-CPI alone after 5 min at 20°C did not cause a significant increase in MLC phosphorylation (12 ± 5 %, n = 5). PKC-1 with 5 µM u-CPI together for 5 min did, as expected, significantly (P < 0·01) increase the levels of MLC phosphorylation, to 32 ± 6 % (n = 4), but to a lower level than the 50 ± 3 % (n = 4) seen at maximum pCa 4·5 (Fig. 8). Higher concentrations of PKC-2 (10 µg ml^-1) in the presence of PS and diC₆ at increased temperature (25°C) for a prolonged duration (30 min) at pCa 6 also significantly (P < 0·03) increased phosphorylation levels of MLC from the control level (13 ± 1 % in the absence of PKC-2; n = 4) to 26 ± 4 % (n = 4) of total MLC, while the force levels were slightly decreased (not shown).

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Figure 8. Effects of PKC-1 and u-CPI on MLC phosphorylation in Triton X-100-demembranated arterial smooth muscle After treatment with 0·1 % Triton X-100, strips were incubated in the relaxing solution at least for 10 min at 20 °C. They were frozen after being partially activated by pCa 6 alone for 15 min (control) or after pCa 6 for 10 min followed by 0·8 µg ml-1 PKC-1, 5 µM u-CPI or PKC-1 plus u-CPI for another 5 min in the pCa 6 solution. The fully activated samples were frozen 5 min after activation by pCa 4·5. Frozen samples were processed for measurement of MLC phosphorylation according to the procedure described in the Methods. *P < 0·01; n = 4-7.

In vitro phosphorylation of CPI-17 and several other isolated smooth muscle proteins by cPKC

To examine the substrate specificity of PKC, we used recombinant PKC wild-type (0·33 µg ml^-1) and MLC, caldesmon, calponin, myosin or CPI-17 as a substrate under the same conditions (see Methods for more details). The CPI-17 was the best substrate for PKC among these proteins; the order of initial velocity of phosphorylation was CPI-17 >> calponin > caldesmon > MLC >> myosin (Fig. 9B). The steady state level of phosphorylation at 40 min for CPI-17, caldesmon, MLC and myosin was, respectively, 3·2, 1·8, 1 and 0·3 mol of phosphate per mol of substrate (Fig. 9A). On the other hand, the level of calponin phosphorylation at 40 min was 4·8 mol per mol and was not saturated. This is consistent with the earlier studies that show the presence of multiple phosphorylation sites of calponin by PKC (Winder & Walsh, 1990; Winder et al. 1993).

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Figure 9. Time course of in vitro phosphorylation of various smooth muscle proteins by PKC (A) and its initial velocity (B) To start the phosphorylation reaction, a high concentration (0·33 µg ml-1) of recombinant PKC wild-type was added to the solution containing 0·2 mM [-32P]ATP and 5 µM substrate, calponin (CaP), caldesmon (CaD), MLC, myosin or CPI-17. Then, the reaction was terminated by mixing the assay solution with 5 % TCA and 1 % sodium pyrophosphate. These time courses are representative of three similar experiments.

In the separate experiments, a much lower concentration (10 ng ml^-1) of cPKC enzyme than that (330 ng ml^-1) used in Fig. 9 was utilized for a short period (3 min) to examine the specificity of Thr-38 phosphorylation as compared with other phosphorylatable sites in CPI-17. The enzyme, depending upon the presence of Ca²⁺, PS and PMA, markedly phosphorylated CPI-17 wild-type but not [T38A]-CPI mutant (Fig. 10), suggesting PKC predominantly phosphorylates CPI-17 at Thr-38.

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Figure 10. Phosphorylation of CPI-17 wild-type and [T38A]-CPI mutant by cPKC Phosphorylation of CPI-17 was initiated by addition of a low concentration (10 ng ml-1) of rat brain cPKC mixture (Upstate Biotechnology, Inc., Lake Placid, NY, USA) to the solution containing 10 mM magnesium diacetate, 1 mM DTT, 0·2 mM Pefabloc, 0·1 mM [-32P]ATP, 25 mM Mops (pH 7·0) and 10 µM CPI-17 wild-type or [T38A]-CPI in the presence or absence of 0·2 mM CaCl2, 0·1 mg ml-1 PS and 0·1 µM PMA. After 3 min incubation at 30 °C, the reaction was terminated with the solution containing 2 % SDS and 1 % 2-mercaptoethanol. Phosphorylation was determined by radioactivity incorporated in the CPI-17 band in the gel followed by SDS-PAGE. WT, wild-type CPI-17; 38A, [T38A] On p-CPI. The values are the means ± S.E.M. of triplicated assays.

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

The major finding in this study is that active PKC enzyme alone cannot directly increase the Ca²⁺ sensitivity of contraction in the calponin-normal, CPI-17-deficient demembranated arterial smooth muscle. However, PKC and CPI-17 together are able to sensitize both contractile force and MLC phosphorylation to Ca²⁺ in the same preparations. This sensitization is comparable to the PKC activator-induced Ca²⁺ sensitization in -toxin-permeabilized preparations. In in vitro experiments, CPI-17 is a much better substrate for PKC than calponin, caldesmon, MLC and myosin. These results suggest that CPI-17 is an endogenous substrate for PKC that evokes a Ca²⁺-independent, MLC phosphorylation-dependent increase in the Ca²⁺ sensitivity. CPI-17 appears to have three sites including Thr-38 that are phosphorylatable by PKC according to the consensus sequence (Eto et al. 1997). It is in fact phosphorylated in vitro to 3 mol of P per mol of CPI-17 (this study). However, that there were no Ca²⁺-sensitizing effects of [T38A]-CPI regardless of phosphorylation by PKC indicates that Thr-38 is the only site for PKC phosphorylation responsible for the contractile Ca²⁺-sensitizing effect.

This study using Triton X-100-demembranated rabbit artery, Inagaki et al. (1987) using saponin-permeabilized rabbit artery, Sutton & Haeberle (1990) using glycerinated porcine artery and Parente et al. (1992) using Triton X-100-demembranated chicken gizzard preparations all found the same result. Although all these preparations appear to have possessed the basic contractile apparatus including calponin (this study) and caldesmon (Nixon et al. 1995), exogenous PKC did not increase the contractile Ca²⁺ sensitivity. Ikebe & Brozovich (1996), however, demonstrated that active PKC enhanced a submaximal contraction at constant Ca²⁺ in -escin-permeabilized single smooth muscle cells. Those results correspond to the findings in this study that CPI-17 is almost totally lost from heavily permeabilized cells and remains to some extent in -escin-permeabilized cells.

Triton X-100 treatment displaces CPI-17 from arterial smooth muscle cells indicating that this protein is cytosolic and soluble or membrane bound, but not associated with myofilaments. The majority of cellular CPI-17 is also diffusible from the receptor/G protein-coupled -escin-permeabilized smooth muscle cells. In such permeabilized cells, since excitatory receptor ligands can still induce Ca²⁺ release from the SR through the receptor/G protein/ phospholipase C pathway (Kobayashi et al. 1989), both surface and SR membranes appear to remain intact to some degree. This suggests that the unphosphorylated form of CPI-17 is soluble but tightly bound to neither lipid membrane nor myofilaments. This is supported by the fact that the primary structure of CPI-17 possesses no consensus sequence patterns for crossing the lipid bilayer or covalent attachment of the lipid membrane (Eto et al. 1997). In contrast, phosphorylated CPI-17 inhibits MLC phosphatase with an EC₅₀ in the nanomolar range (Eto et al. 1997; Li et al. 1998), suggesting tight binding to the thick filament-associated phosphatase. The mechanisms by which active PKC and/or other kinases recognize cytosolic CPI-17 and the resultant phosphorylated CPI-17 is targeted to MLC phosphatase associated with thick filaments are of particular interest.

PKC isoform(s) responsible for phosphorylating in situ CPI-17 have not been yet identified. In in vitro experiments, a PKC fraction isolated from porcine brain that mainly contained the conventional isoforms was used for phosphorylating isolated native and recombinant CPI-17 (Eto et al. 1995, 1997; Li et al. 1998). Eto et al. (1995) isolated from porcine aorta a protein kinase that strongly phosphorylated CPI-17 and was inhibited by PKC-IP but was not recognized by a particular anti-conventional PKC antibody. A major component of the aorta CPI-17-phosphorylating protein kinase fraction has been recently identified as a novel class of PKC -isoform (M. Eto, unpublished observations). In this study, conventional PKC and novel PKC were utilized for phosphorylating recombinant CPI-17 proteins. CPI-17 also appears to be phosphorylated in the Triton X-100 demembranated rabbit arterial smooth muscle by the in situ protein kinase(s) that is pharmacologically identified as the conventional PKC (see below for more discussion). These results suggest that at least conventional and novel and isoforms of PKC can phosphorylate CPI-17 at Thr-38. However, identification of the PKC isozyme(s) phosphorylating in situ CPI-17 and the possibility of other protein kinases and mechanisms other than phosphorylation being involved in activation remain to be studied.

Unphosphorylated CPI-17 has a slight but significant potentiating effect on contractile force at constant Ca²⁺ in Triton X-100-demembranated and -escin-permeabilized strips (this study; Li et al. 1998). Since purified u-CPI induces a weak but significant direct inhibition of phosphatase activity in vitro (Eto et al. 1997; Li et al. 1998), the u-CPI-induced contractile effect was previously thought to be due to a direct inhibitory effect of u-CPI on in situ MLC phosphatase. However, the u-CPI-induced contractile Ca²⁺ sensitization was totally inhibited by selective inhibitors of conventional PKC isoforms, indicating involvement of PKC phosphorylation. It is possible that PKC, although found in much smaller amounts than that in intact tissues, remains active in the strips after Triton X-100 or -escin treatment. The hinge region between the kinase and regulatory domains has been demonstrated to be subjected to proteolytic cleavage to produce a constitutively active fragment protein kinase M (Kishimoto et al. 1983). Although the presence of protease inhibitors throughout the experiments did not affect the u-CPI-induced contraction and such PKC fragments without regulatory domain would hardly remain in the demembranated strips, the possibility of PKM being responsible for the phosphorylation of u-CPI cannot be totally excluded. Autophosphorylation (Mochly-Rose & Koshland, 1987) or oxidation of PKC (Gopalakrishna & Anderson, 1989) has also been demonstrated to increase the kinase activity in vitro and might be responsible for the u-CPI-induced contraction.

Calponin, a smooth muscle-specific, thin filament-associated protein, has been proposed as a regulatory protein in smooth muscle. Calponin inhibits actomyosin ATPase (Winder & Walsh, 1990; Abe et al. 1990) and cross-bridge cycling in in vitro motility assays (Shrinsky et al. 1992; Haeberle, 1994). Its phosphorylation by PKC or Ca²⁺/calmodulin-dependent protein kinase II reverses this inhibition, suggesting that calponin phosphorylation can evoke Ca²⁺-independent, MLC phosphorylation-independent contraction (Winder & Walsh, 1990; Rokolya et al. 1994). Several reports (see review by Singer, 1996; and Horowitz et al. 1996) have documented that PKC activators evoked a MLC phosphorylation-independent contraction. However, we along with others found a significant increase in MLC phosphorylation at Ser-19 during same PKC activator-induced, Ca²⁺-independent contraction in both intact and permeabilized arterial smooth muscles (Itoh et al. 1993; Masuo et al. 1994; Gailly et al. 1997). Furthermore, the question of whether phosphorylation of in situ calponin occurs during contractile activation is still controversial. Several studies (Allen & Walsh, 1994; Mino et al. 1995; Rokolya et al. 1996; Pohl et al. 1997) found increased levels of the phosphorylation upon stimulation while others (Gimona et al. 1992; Barany & Barany, 1993; Adam et al. 1995) could not confirm this finding. This study has confirmed and further extended the previous results (Inagaki et al. 1987; Sutton & Haeberle, 1990; Parente et al. 1992); the active PKC enzymes did not potentiate a submaximal contraction at constant Ca²⁺ in the calponin-containing demembranated smooth muscle.

There are, however, some possibilities for the failure to observe any Ca²⁺-sensitizing effect of exogenously applied PKC enzymes on the demembranated arterial strips in which calponin is present. First of all, exogenous PKC might not be accessible to in situ calponin associated with thin filaments. This, however, is unlikely because a large molecule 150 kDa IgG did effect the contractile Ca²⁺ sensitization in the same type of arterial preparations through binding to MLC kinase associated with contractile filaments. In in vitro conditions, several PKC isoforms (PKC-2 and PKC in this study) need for their activation to bind to large PS micelles of submicrometre diameters, which might be too large to penetrate into myofibrils. This is also not the case in this study because PKC-1, which does not have to be anchored to the PS micelles, had no significant effect on contractile force levels, and PKC-2, requiring the micelles for its activation, did significantly phosphorylate poor substrate in situ myosin during prolonged incubation and at higher temperature. This suggests that these enzymes can penetrate into myofibrils but have no Ca²⁺-sensitizing effect. Whether or not phosphorylation of in situ calponin occurs under these conditions, however, remains to be determined. Secondly, Parker et al. (1998) have proposed a new hypothesis that PKC-induced MLC phosphorylation-independent contractile activation occurred with a translocation of calponin from -smooth muscle actin filaments in the contractile domain to peripheral cytoskeletal -actin filaments through an unknown mechanism. A possibility, which cannot be discarded, is that in the demembranated strips, calponin might already be somehow translocated to -actin and thus its inhibition of cross-bridge cycling already removed. In addition, unknown cofactor(s) necessary to the calponin translocation might be extracted or deformed by the detergent treatment.

Caldesmon is the other thin filament-associated regulatory protein which binds to myosin and inhibits actomyosin ATPase and thin filament movement in in vitro motility assays (Sobue & Sellers, 1991; Shrinsky et al. 1992). This in vitro inhibition is removed by phosphorylation of caldesmon by several protein kinases including PKC. Adam et al. (1992), however, showed increased caldesmon phosphorylation at specific proline-directed serine residues during prolonged contractions induced by PKC activator and KCl. Mitogen-activated protein kinase (MAPK) has thus been suggested to be the endogenous caldesmon kinase. Since MAPK requires dual phosphorylation at threonine and tyrosine residues for full activation and endogenous MAPK appears to be depleted by Triton X-100 treatment (Nixon et al. 1995), the signalling pathway involving caldesmon cannot be activated by exogenously applied PKC alone in the demembranated smooth muscle. Although caldesmon may be involved in the development and maintenance of smooth muscle contraction, the in situ phosphorylation of caldesmon by MAPK has been shown to cause no significant effect on the contractile Ca²⁺ sensitization in permeabilized smooth muscle (Nixon et al. 1995).

In summary, Triton X-100 treatment eliminates the Ca²⁺-sensitizing effect of PKC activators on arterial smooth muscle contraction while simultaneously depleting CPI-17 and PKC from cells. Yet Triton X-100 does not significantly affect the contents of either -smooth muscle actin or calponin. Using this CPI-17-deficient, calponin-normal arterial smooth muscle model, the Ca²⁺ sensitization of contraction and MLC phosphorylation is evoked by replenishment of exogenous PKC and CPI-17 together, but not by PKC alone. Mutant [T38A]-CPI, however, does not support the PKC-induced Ca²⁺ sensitization. These results suggest that PKC activated by phorbol ester and DAG phosphorylates cytosolic CPI-17 at Thr-38 and the resultant phosphorylation provokes an inhibition of MLC phosphatase to increase the Ca²⁺ sensitivity of MLC phosphorylation and contraction in smooth muscle.

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Acknowledgements

The authors are grateful to Matthew R. Lee for his valuable discussion on the manuscript and Terence P. Woodsome for his useful comments. This work was supported by NIH Grant HL51824 and a grant from the American Heart Association to T.K., and by NIH grants HL60831 and HL61426 to M.I.

Corresponding author

T. Kitazawa: Department of Physiology and Biophysics, Georgetown University Medical Center, 3900 Reservoir Road, N. W., Washington, DC 20007, USA.

Email: tkitaz01{at}gusun.georgetown.edu

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