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Am J Physiol Cell Physiol 294: C564-C571, 2008. First published December 19, 2007; doi:10.1152/ajpcell.00285.2007
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VASCULAR BIOLOGY

Uterine artery myosin phosphatase isoform switching and increased sensitivity to SNP in a rat L-NAME model of hypertension of pregnancy

Yuan Lu,1 Haiying Zhang,1 Natalia Gokina,2 Maurizio Mandala,3 Osamu Sato,4 Mitsuo Ikebe,4 George Osol,2 and Steven A. Fisher1

1Department of Medicine (Cardiology), Case Western Reserve School of Medicine, Cleveland, Ohio; 2Department of Obstetrics and Gynecology, College of Medicine, University of Vermont, Burlington, Vermont; 3Department of Cell Biology, University of Calabria, Cosenza, Italy; and 4Department of Physiology, University of Massachusetts Medical School, Worcester, Massachusetts

Submitted 20 July 2007 ; accepted in final form 7 January 2008


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Dramatic and vascular bed-specific hemodynamic changes occur in pregnancy and hypertension of pregnancy (HtP). Because myosin phosphatase (MP) is the primary effector of smooth muscle relaxation and a key target of signaling pathways that regulate vascular tone, we hypothesized that MP expression would be altered in these conditions. The abundance of the targeting/regulatory subunit of MP (MYPT1) mRNA and protein was increased 1.7- to 2.0-fold specifically in the uterine arteries (UAs) of late-pregnant rats without isoform switching. In a model of HtP in which nitric oxide (NO) synthesis is blocked by the chronic administration of N{omega}-nitro-L-arginine methyl ester, MYPT1 was downregulated and switched to the splice variant isoform that codes for the COOH-terminal leucine zipper motif. This was associated with increased sensitivity of the main UA and its subbranches to the vasorelaxant effects of the NO donor drug sodium nitroprusside. This difference was abolished by pretreatment with the phosphatase inhibitor tautomycetin. The sensitivity of relaxation to the NO second messenger cGMP was also increased under calcium-clamp conditions in permeabilized UAs, indicating heightened activation of MP. The changes in MP expression in HtP were largely prevented by treatment with the antihypertensive medicine hydralazine. We propose that MYPT1 isoform switching is an adaptive response to reduce vascular resistance and maintain uterine blood flow in the setting of hypertension-triggered inward remodeling of the UAs in hypertension of pregnancy.

smooth muscle; gene expression; guanosine 3',5'-cyclic monophosphate; contractility; signaling; N{omega}-nitro-L-arginine methyl ester; sodium nitroprusside

A MULTITUDE OF CHANGES OCCUR in the circulation of mammals during normal pregnancy. One critical adaptation is the fall in the uterine vascular resistance that allows for the 15- to 20-fold increase in uterine blood flow needed to nourish the growing fetus. Approximately 5–10% of human pregnancies are complicated by hypertensive disorders of pregnancy leading to increased perinatal morbidity and mortality for the mother and fetus (12, 22). These disorders include pregnancy-induced hypertension, chronic hypertension, preeclampsia, and eclampsia. A number of studies have suggested that the pregnancy-related reduction in vascular resistance, particularly in the uterine vascular bed, is in part due to increased synthesis and activity of endothelium-derived vasodilators such as nitric oxide (NO) and prostacyclin (reviewed in Ref. 1). Similarly, it has been proposed that endothelial dysfunction, manifest as defective release/activity of the vasodilators, as well as increased release and activity of vasoconstrictors such as angiotensin II, thromboxane, and endothelin, may contribute to the elevated vascular resistance in hypertension of pregnancy (HtP; reviewed in Ref. 9).

Alternatively, or in addition, the changes in vascular resistance in HtP could be due to altered reactivity of the small resistance arterial/arteriolar smooth muscle to vasodilator and vasoconstrictor signals. A number of models have been developed to study the mechanistic basis of HtP in the rat, including surgically induced uteroplacental vascular insufficiency and inhibition of NO synthesis (NOS) with the chronic administration of arginine analogs such as N{omega}-nitro-L-arginine methyl ester (L-NAME; reviewed in Ref. 21). These studies have shown pathological remodeling of systemic and uterine resistance vessels in these models.

A number of studies using the model of NOS inhibition-induced hypertension in pregnant or nonpregnant animals have demonstrated changes in vascular reactivity that favor constriction by increased RhoA, PKC, and calcium signaling (2, 8, 13). Information regarding uterine artery (UA) reactivity to vasodilators in HtP is quite limited, with only one report to date (26). In that study, which used ring-mounted segments of the rat main UA, there were no changes in maximal relaxation to ACh or sodium nitroprusside (SNP) or in constrictor responses to norepinephrine and serotonin; however, sensitivity to SNP was significantly enhanced in the L-NAME-treated group vs. pregnant controls. There have not been any studies of the smaller, mesometrial resistance vessels that play an important role in the regulation of myometrial and uteroplacental blood flow (15).

We have been studying myosin phosphatase (MP), which by dephosphorylating myosin light chain is the primary effector of smooth muscle relaxation. MP is also a key target of vasoconstrictor and vasodilator signals, which by inhibiting or enhancing its activity, respectively, increase or decrease the force produced at any given concentration of activating calcium (reviewed in Ref. 5). MP is a heterotrimer composed of catalytic (PP1c), targeting/regulatory (MYPT1), and 21-kDa (M21) subunits. A leucine zipper (LZ) motif present at the COOH terminus of MYPT1 mediates its dimerization with the cGMP-dependent protein kinase, and it is required for NO/cGMP-mediated activation of MP and calcium desensitization of force production (7, 10, 25). Isoforms of MYPT1 that contain or lack the COOH-terminal LZ are generated by the tissue-specific and developmentally regulated splicing of a 31-nt alternative exon. Inclusion of this 3' alternative exon shifts the reading frame, resulting in a premature stop codon and a MYPT1 isoform that lacks the COOH-terminal LZ sequence. On the basis of studies in other models, we have proposed that the regulated expression of the MYPT1 LZ isoforms serves as a way for smooth muscle to fine tune its response to NO/cGMP signaling in development and in diseases in which blood flow or pressure is altered (10, 19, 20, 28). In the models of altered blood flow/pressure, we have observed a partial or complete switch in MYPT1 expression to the LZ-positive (LZ+) isoform associated with increased sensitivity to NO/cGMP signaling. This led us to hypothesize that switching of MYPT1 to the LZ+ isoform associated with increased sensitivity to NO/cGMP signaling may occur in pregnancy and HtP. To test this hypothesis, we examined the expression of MP and vasodilator responses in uterine and first-order mesenteric arteries (MAs) during normal rat pregnancy and the L-NAME model of HtP.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals and reagents. Ten-week-old Sprague-Dawley rats were purchased from Charles River Laboratories and housed in the animal facility for 2 wk before breeding or initiation of treatment. All animals were housed in an environmentally controlled vivarium and had free access to food and water. All experimental protocols were approved by the Animal Care and Use Committee at Case Western Reserve University. A separate group of age-matched Sprague-Dawley rats underwent the same treatments at the University of Vermont, with approval from the institutional Animal Care and Use Committee; these animals were used for small artery reactivity experiments under isobaric pressurized conditions, as described below. All chemicals except where noted were purchased from Sigma.

Model of HtP. Rats were divided into five groups as follows: group 1, virgin; group 2, pregnant; group 3, H + P: pregnant + L-NAME; group 4, HtP+Hyd: pregnant + L-NAME + hydralazine; and group 5, HtP+captopril: pregnant + L-NAME + captopril. A total of 9–11 rats were used in each group except group 5, for which n = 4. L-NAME was administrated from the 7th day of pregnancy at 0.5 g/l in drinking water for 12–14 days. Rat daily water consumption was ~100 ml·day–1·kg body wt–1; the dose of L-NAME was ~50 mg·day–1·kg body wt–1. In the HtP+Hyd and HtP+captopril groups, after 4 days of treatment with L-NAME, hydralazine or captopril was administrated in drinking water at 0.25 and 0.15 g/l, the dose of which is ~25 and ~15 mg·day–1·kg body wt–1, respectively. With the use of the tail-cuff method, systolic blood pressure (SBP) measurements were taken at the same time of day on days 3, 7, 10, 13, 15, 18, and 20 of pregnancy with a Programmed Electro-Sphygmomanometer (PE-300, Narco Bio-systems International Biomedical). After two blood pressure measurements that were used as an adaptation period, four readings were taken and averaged for each measurement.

Analysis of MP expression and activity. Rats were euthanized after isoflurane inhalational anesthesia by thoracotomy and ventricular transection. The main UAs and first-order MAs ({Phi} ~ 100–400 µm) and other reference vessels, aorta, and portal vein were dissected in ice-cold physiological saline solution (PSS) containing (in mM) 119 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 25 NaHCO3, 1.2 NaH2PO4, 0.027 EDTA, and 5.5 glucose, stripped of adventitia and frozen in liquid nitrogen. Tissues were processed for RNA (TRIzol, Invitrogen, Carlsbad, CA), protein (10% SDS homogenization buffer) or enzymatic activity by standard methods as previously described (19, 20).

RT-PCR and real-time PCR. Total RNA was treated with Turbo DNA-free (Ambion) to degrade contaminating DNA and analyzed for MYPT1 abundance and isoforms as previously described (20). In brief, 1 µg RNA was reverse transcribed by using an oligo-dT primer and 1 µl (200 units) Superscript RT (Invitrogen). A single set of oligonucleotide primers that bracket the MYPT1 3' alternative exon was used to amplify both isoforms in a single reaction. PCR was performed with 1 µl cDNA in a volume of 50 µl and with cycles of 94°-55°-72°C for 30–30-30 s. PCR products were separated by 8% PAGE, and exon-included to exon-excluded ratios were determined with a Storm 860 Imager and ImageQuant software (version 1.2, Molecular Dynamics) as described previously (20). Real-time PCR primers were designed with the Primer3 program. Basic Local Alignment Search Tool (BLAST) analysis was performed to confirm the specificity of primers. The following two sets of oligonucleotide primers were used: {alpha}-actin, 5'-GCTCTGGTGTGTGACAATGG-3', 5'-AACCATCACTCCCTGGTGTC-3'; and MYPT1, 5'-CGAAGCGGAGACAGATAAGA-3', 5'-GTTGTCACAGCGGCAGGA-3'. {alpha}-Actin was used as internal reference for normalization. Fold changes of MYPT1 transcripts were calculated as 2Formula, where Ct is threshold cycle.

Western blot analysis. Total protein was isolated from different vessels, and protein abundance was measured by Western blotting and band densitometry as previously described (20). Briefly, vessels were homogenized in 200 µl lysis buffer [125 mM Tris·HCl, pH 6.8, 20% sucrose, 10% SDS, and 1% proteinase inhibitor cocktail (Sigma, St. Louis, MO)]. Protein (8 µg) was separated on 3–8% NuPAGE Tris-acetate gels (Invitrogen) at 50 mA for 1.5 h and was transferred to polyvinylidene difluoride membranes by standard methods at 300 mA for 1.5 h. Primary antibodies used included the following: MYPT1 (rabbit polyclonal IgG M130, Abcam, ab24670, 1:3,000), COOH-terminal LZ+ of MYPT1 (20) (rabbit polyclonal IgG; 1:3,000), MLCK (mouse monoclonal IgG, Sigma, 1:5,000), {alpha}- actin (mouse monoclonal IgG, Sigma, clone 1A4, 1:10,000).The semiquantitative nature of the assay was established by the observation of a linear relationship between amount of protein loaded from 2–15 µg and the measurement (data not shown).

MP activity was measured as previously described with minor modifications (20). In brief, pairs of UAs were removed from virgin, pregnant, or HtP rats (n = 3 each) and were treated with 10–7 M SNP for 5 min, protein was extracted, and protein concentrations were normalized to 2.0 mg/ml. The lysates were incubated with 0.1 µM 32P-labeled smooth muscle myosin at 25°C. The assay was terminated by the addition of TCA to 5%. The samples were sedimented, and the radioactivity in the supernatant was determined by Cerenkov counting.

Large and small UA relaxant responses to SNP. The contractile responses of the main UA from paired control and experimental rats were measured in parallel in a four- chamber myograph (DMT model 610M, Danish Myotechnology) as previously described (28) with minor modifications. The midpoints of the main UAs were isolated in ice-cold PSS (2 mm in length), mounted in PSS, and bubbled for 30 min while the temperature was allowed to increase gradually to 37°C. Normalization was done according to Mulvany's method (16). In brief, the internal circumferences of the vessels were set to 90% of that when transmural pressure reached 50 mmHg. Two priming doses of phenylephrine (PE; 10–5 M) were added to make sure that the maximal contraction was reproducible. The vessels were pretreated with the NO inhibitor L-NAME at 10–4 M for 30 min to block endogenous NO production. Vessels were activated with PE to give 80% of maximal force. After force reached steady state, the vessel was exposed to incremental concentrations of the NO donor drug SNP (10–9 to 10–4.5 M) while force was continuously recorded. At the end of the protocol, PSS was added to demonstrate complete relaxation.

The same experimental protocol was used on UAs from virgin, pregnant, and HtP rats (n = 3 each) pretreated with tautomycetin, a selective type 1 phosphatase inhibitor (14). Uterine artery segments from virgin, pregnant, and HtP groups were preincubated with 1 µM tautomycetin for 20 min. The vessel segments were then challenged with 10–5 M PE followed by incremental concentrations of SNP (10–9 to 10–4 M). The change in force was continuously recorded and analyzed.

Another set of main UAs was permeabilized with 800 U/ml {alpha}-toxin as previously described (3), except that vessels were maintained at 37°C. Force was activated with a submaximal concentration of calcium (10–6 M). 8-Bromo-cGMP (8-Br-cGMP) was added to assess the effect of cGMP on calcium sensitivity of force production. Time vs. force tracings were recorded, and percent change in steady-state force and rate of relaxation were calculated. Results are reported as means ± SD percent change in force. At the end of the protocol, PSS was added to demonstrate complete relaxation.

Additional reactivity studies were performed with uterine mesometrial radial arteries to determine whether changes in reactivity were present in the preplacental radial arteries that contribute significantly to uterine vascular resistance and blood flow regulation (15). The segments of uterine radial arteries (diameters ranged from 80 to 250 µm at 50 mmHg) were dissected free of surrounding connective tissue and were mounted on two glass cannulae within the chamber of a specialized arteriograph (Living Systems Instrumentation, Burlington, VT). Following equilibration at 50 mmHg in a solution of HEPES buffer, vessels were preconstricted with PE to achieve 40–60% reductions in lumen diameter. SNP (10–9 to 10–4 M) was then added in increasing concentrations until maximal dilation was achieved, and changes in lumen diameter were recorded with a video dimension analyzer (Living Systems Instrumentation) and a multichannel software program (Ionoptix). Data were then plotted relative to maximal dilation as a function of SNP concentration. The addition of diltiazem (10–5 M) and papaverine (10–4 M) at the end of each experiment was used for the calculation of efficacy (%maximal dilation). In all of the studies of vascular reactivity, only those vessels that showed normal force generation and complete relaxation were included in the analysis.

Statistical analysis. Data were analyzed and expressed as means ± SD. Data were compared with one-way ANOVA and Tukey posttest. Differences were considered statistically significant at a value of P < 0.05. For the reactivity studies, the EC50 values represent the negative logarithm of the drug concentration required to produce 50% of the maximal response (dilation).


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
L-NAME-induced HtP rat model. The SBP of pregnant rats was elevated after 4 days of treatment with 0.5 g/l of L-NAME in the drinking water (H + P: 149 ± 19 mmHg, P < 0.01 vs. pregnant: 109 ± 7 mmHg). The SBP remained elevated with continued L-NAME treatment for the remainder of the pregnancy (Fig. 1). The addition of the antihypertensive medicine hydralazine 0.25 g/l in the drinking water after 4 days of L-NAME treatment, along with continued L-NAME treatment, significantly reversed the elevation in BP (HtP+Hyd: 128 ± 14 mmHg, P < 0.01 vs. HtP: 160 ± 20 mmHg, P > 0.05 vs. pregnant: 109 ± 4 mmHg). The SBP was not different between pregnant and virgin rats (Fig. 1).


Figure 1
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  		Fig. 1. Average systolic blood pressures measured with the tail-cuff method. N{omega}-nitro-L-arginine methyl ester (L-NAME) increased systolic blood pressure by ~30%, and hydralazine reversed the elevated blood pressure. Vgn, virgin; P, pregnant; HtP, hypertension of pregnancy; HtP + Hyd, HtP + hydralazine treatment. ##P < 0.01 vs. P; **P < 0.01 vs. HtP.

 
MYPT1 isoform switching in HtP. In virgin female rats, the ratio of MYPT1 3' alternative exon-excluded to exon-included in the UA as determined by RT-PCR was 46/54 (n = 7; Fig. 2), coding for a nearly equal mix of LZ+ and LZ-negative (LZ–) isoforms. Near the end of a normal pregnancy (days 20-21 of a 22-day gestation), the MYPT1 3' alternative exon-excluded to exon-included ratio in the UA was unchanged (pregnant: 49/51, n = 9; P > 0.05 vs. virgin). In HtP, after 14 days of L-NAME treatment (pregnancy days 7-21), there was a significant shift of MYPT1 to exon-exclusion coding for the LZ+ isoform (MYPT1 3' alternative exon-exclusion to exon-inclusion ratio 74/26, n = 11; P < 0.001 vs. pregnant). There was no change in the MYPT1 isoform ratios after only 4 days of treatment with L-NAME at 0.5 g/l, although the BP was significantly elevated at this time point (data not shown). Treatment with the antihypertensive medicine hydralazine normalized the MYPT1 isoform ratio (HtP+ Hyd: 50/50, n = 8, P < 0.001 vs. HtP; P > 0.05 vs. pregnant). Treatment of the HtP rats with the antihypertensive medicine captopril also normalized the UA MYPT1 isoform ratios (HtP+captopril: 46/54 ± 8, n = 4; P < 0.01 vs. HtP, P > 0.05 vs. pregnant).The changes in MYPT1 isoforms ratios in HtP were specific to UAs, because there were no changes in the systemic mesenteric resistance artery (MA1) (virgin: 16/84, n = 6; pregnant: 19/81, n = 6; HtP: 22/78, n = 4; P > 0.05). There were no changes in MYPT1 isoforms in the tonic smooth muscle of the aorta, which is predominately MYPT1 LZ+, or in the phasic smooth muscle of the portal vein, which is predominately MYPT1 LZ– (data not shown).


Figure 2
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  		Fig. 2. Switching in uterine artery targeting/regulatory subunit of myosin phosphatase (MYPT1) 3' splice variant isoform ratios as measured by RT-PCR. Ratios of exon-excluded to exon-included MYPT1 transcripts were measured with a single-set Cy3-labeled oligonucleotide PCR as described in METHODS. Shown is a representative gel and the quantification from 7–11 independent samples. %Exon exclusion represents the signal from the exon-excluded band divided by the total signal from the exon-excluded and exon-included bands. Data are presented as means ± SD. ##P < 0.01 vs. P; **P < 0.01 vs. HtP.

 
We used a previously characterized antibody that specifically recognizes the COOH-terminal LZ motif present in MYPT family members MYPT1, p85, and M21 (described in METHODS and in Ref. 20) to examine the isoform expression at the level of the protein. The ratio of the MYPT1 LZ:MYPT1 signal in the UAs was not different between pregnant and virgin rats (Fig. 3). This ratio was increased by ~1.5-fold in the HtP rats (Fig. 3A), and was normalized by hydralazine treatment, all consistent with the isoform switching observed by RT-PCR. Also evident in the Western blot in Fig. 3A is the increase in HtP in the LZ signal of MYPT1 relative to the related family member p85, which shares the same COOH-terminal LZ sequence.


Figure 3
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  		Fig. 3. Changes in uterine artery MYPT1 protein expression as measured by Western blot analysis. A: an antibody was used that specifically detects the leucine zipper (LZ) sequence present in MYPT family members MYPT1 and p85. B: blots were probed with a rabbit polyclonal antibody that recognizes all isoforms of MYPT1. The blots were reprobed with an antibody to smooth muscle {alpha}-actin as an internal control. In A, MYPT1 LZ signal was normalized to total MYPT1. In B, total MYPT1 was normalized to {alpha}-actin, which was invariant. All data were normalized to the Vgn group. Data are presented as means ± SD. ++P < 0.01 vs. Vgn, ##P < 0.01 vs. P, **P < 0.01 vs. HtP.

 
Changes in MYPT1 abundance in pregnancy and HtP. Total MYPT1 protein was increased by approximately twofold in the UAs of pregnant rats as compared with virgin rats (Fig. 3B), normalized to the smooth muscle {alpha}-actin signal, which was invariant. MYPT1 protein decreased by 55% in the HtP UAs as compared with the normal pregnant UAs. Treatment with hydralazine significantly attenuated the decline in MYPT1 protein abundance observed in HtP. There were no changes in MYPT1 abundance in the MA1, aorta, or portal vein in any of these groups (data not shown).

Total (nonisoform specific) MYPT1 transcript abundance as measured by real-time PCR was increased by 1.8-fold in the pregnant UAs as compared with the virgin UAs (Fig. 4). All signals were normalized to smooth muscle {alpha}-actin, which did not differ between groups. The MYPT1 mRNA was decreased by 65% in the HtP UAs as compared with the pregnant UAs. Treatment with hydralazine partially reversed the decline in MYPT1 mRNA observed in HtP. Thus the changes in MYPT1 mRNA and protein levels were closely correlated in these different groups.


Figure 4
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  		Fig. 4. Changes in uterine artery MYPT1 transcript levels as measured by real-time PCR. Total MYPT1 transcripts were measured by real-time PCR as described in METHODS and were normalized to values for smooth muscle {alpha}-actin, which were not different between groups. All data were normalized to the Vgn group. Data are presented as means ± SD. +P < 0.05 vs. Vgn, ##P < 0.01 vs. P, **P < 0.01 vs. HtP.

 
Relaxation responses to SNP and cGMP. Given the role of MP as the primary mediator of smooth muscle relaxation, and a target of signaling pathways that regulate vascular tone, the changes in MYPT1 expression in pregnancy and HtP supported the hypothesis that UA relaxant responses would be altered. Isolated main UAs were preconstricted with the {alpha}- adrenergic agonist PE. The normalized dose-response curves and EC50 for the contractile response to PE did not differ between groups (data not shown). The maximum force development differed by up to 20% between groups. Media thickness also varied between groups, confounding comparisons of maximum force. After PE preconstriction (10–5 M), there was no significant difference between virgin and pregnant UAs in the dose-response of relaxation to 10–9~10–5 M SNP (Fig. 5A). At the highest concentration tested, 10–4.5 M, the pregnant UAs showed a greater relaxation (80.8 ± 6.4% vs. 64.6 ± 2.9%, n = 5, P < 0.01, n = 4 each). The UAs from the HtP rats were significantly more sensitive than the pregnant UAs to SNP (HtP: EC50 = 7.77 ± 0.20, n = 7 vs. P: EC50 = 7.06 ± 0.18, n = 5, P < 0.01; Fig. 5A) with no difference in the maximal relaxation. Tautomycetin, a phosphatase inhibitor with selectivity for type 1 phosphatases (14), was used to test the role of phosphatases in the increased sensitivity of the HtP UAs to SNP. After pretreatment with 10–6 M tautomycetin, the differences in the sensitivity to SNP among the groups were abolished, and the dose-response curves of all groups were shifted rightward (Fig. 5B). Maximal relaxation to SNP was also significantly reduced in all three groups.


Figure 5
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  		Fig. 5. Sodium nitroprusside (SNP) dose-response relationships of the main uterine artery and its subbranches. A: main uterine arteries were mounted on a wire myograph and force continuously recorded. Arteries were preconstricted with 10–5 M phenylephrine and then exposed to increasing concentrations of the NO donor SNP. The data are plotted as percent relaxation from maximum force. n = 4–7 in each group. B: the main uterine arteries were treated as in A but were pretreated with the phosphatase inhibitor tautomycetin (10–6 M). n = 3. C: the smaller uterine mesometrial radial arteries were studied in a pressurized isobaric system. Arteries were preconstricted with phenylephrine to achieve a 40–60% reduction in lumen diameter before the administration of SNP. The data are plotted as the percent change in lumen diameter relative to maximal relaxation. (n = 6 each). All data are presented as means ± SD. ##P < 0.01 vs. P, ++P < 0.01, P vs. Vgn, #P < 0.05 vs. P. –lgEC50, –log10 of the concentration of SNP that causes 50% of the maximal effect.

 
Because resistance to blood flow to the uterus is predominately at the level of the smaller radial arteries (15), we next sought to determine whether these vessels would also show increased sensitivity to SNP in this model of HtP. Reactivity of the radial UAs (~200 µm diameter) was studied in a pressurized system in vitro. After PE preconstriction, vessels from the HtP group were significantly more sensitive to the relaxant effects of SNP than the pregnant controls (EC50 values: HtP, 8.85 ± 0.43, n = 6; pregnant, 6.55 ± 0.45, n = 6; P < 0.05; Fig. 5C). The extent of relaxation relative to maximal dilation was similar in all three groups, with 94–96% dilation observed in vessels from each treatment group.

NO signaling through cGMP may cause vasorelaxation through reduction of calcium flux or through reduced calcium sensitivity of the myofilaments (reviewed in Ref. 6). A switch to the MYPT1 LZ+ isoform is proposed to render MP sensitive to activation by the cGMP-dependent protein kinase, thereby facilitating smooth muscle relaxation in the presence of activating calcium, i.e., calcium de-sensitization of force production (reviewed in Ref. 5). To determine if the increased sensitivity of the HtP UA to SNP may be due to greater cGMP-mediated calcium de-sensitization (MP activation) the main UAs were permeabilized and relaxation to the non-hydrolyzable analog of cGMP, 8-Br-cGMP, measured at fixed calcium concentrations. At calcium concentrations that caused sub-maximal force production (10–6 M), the HtP UA was significantly more sensitive to the relaxing effect of 8-Br-cGMP as compared with the pregnant UA (EC50 values: HtP, 7.46 ± 0.38, n = 3; pregnant, 6.23 ± 0.17, n = 4, P < 0.01; Fig. 6). There was no difference in the maximal relaxation to the cGMP analog in pregnant and HtP UAs.


Figure 6
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  		Fig. 6. 8-Bromo-cGMP (8-Br-cGMP) dose-response relationships of permeabilized main uterine arteries. {alpha}-Toxin-permeabilized uterine arteries were contracted with 1 µM calcium, and the relaxation responses to cumulative concentrations of 8-Br-cGMP were measured. The data are plotted as percent relaxation from maximum force. #P < 0.05 vs. P.

 
MP activities were measured in virgin, pregnant, and HtP tissue homogenates after exposure of the UAs to 10–7 M SNP. These activities reflect both MP basal activity as well as activation by SNP. The rate of myosin dephosphorylation was highest in pregnant, intermediate in HtP, and lowest in virgin UAs (Table 1). When normalized to relative expression of MYPT1, the activity was highest in HtP, intermediate in virgin, and lowest in pregnant. These were trends that did not quite reach statistical significance (n = 3 in each group, P = 0.07 by ANOVA) owing to the small sample size. The higher SNP- stimulated MP-specific activity in HtP UAs supports the increased sensitivity of HtP to SNP and cGMP-mediated relaxation and calcium desensitization as compared with pregnant UAs, even though the latter expresses twofold more MYPT1.


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  		Table 1. Myosin phosphatase activity

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we have shown that the expression of the MYPT1 is increased specifically in the UAs in pregnancy without any switch in isoform expression. In HtP induced by chronic inhibition of NOS, MP expression is decreased along with a switch to the MYPT1 LZ+ isoform, specifically in the UAs. This is associated with increased sensitivity of the vessel to the nitrovasodilator SNP and its second messenger cGMP, and this difference is abolished by the phosphatase inhibitor tautomycetin. The changes in MP expression are normalized by treatment with the antihypertensive medicines hydralazine and captopril. These results suggest that the regulated expression of the MP is an important determinant of organ-specific alterations in vascular function that occur in pregnancy and HtP.

In normal pregnancy, there was no change in the MYPT1 isoforms expressed either in the main UA or in the reference systemic MA1. This is consistent with the vascular reactivity data in this and other studies showing no change in the sensitivity of these vessels to the NO donor drug SNP (18; reviewed in Ref. 24). Thus the substantial fall in the resistance of the uterine vascular bed and the 15- to 20-fold increase in blood flow do not appear to be due to increased sensitivity of the vascular smooth muscle to signaling through the NO/cGMP pathway. There was a significant shift in MYPT1 isoforms in the L-NAME model of HtP. That this is a specific response to the increased BP induced by L-NAME administration is suggested by the absence of a change in MP expression after 4 days of NOS inhibition, thus suggesting that inhibition of NOS by itself is insufficient to induce this effect. The change in MP expression was prevented by the coadministration of the antihypertensive medicines hydralazine or captopril. Thus, in a setting of continuous L-NAME-induced inhibition of NOS, the treatment with the antihypertensive medicine prevented the switch in MYPT1 isoforms. This further supports the contention that loss of NO signaling in and of itself is not sufficient to trigger a change in MYPT1 isoform expression in the UAs. In contrast, there was no change in MYPT1 isoform expression in the MA1 in L-NAME-induced hypertension of pregnancy or in pregnancy itself. This contrasts with two other models we have studied: the portal vein ligature model of portal hypertension, and the second-order MA ligation inducing chronic high and low flow states in the upstream MA1. In each of these models, there is a partial or complete shift of the MA1 smooth muscle to the MYPT1 LZ+ isoform associated with increased sensitivity to NO/cGMP signaling (20, 28). One possible explanation for the absence of MYPT1 isoform switching in the MA1 in L-NAME-induced hypertension, and in the UA in pregnancy without hypertension, is that a combination of increased transmural pressure and altered flow is required. It is also possible that the uterine and mesenteric arterial smooth muscle may respond differently to sustained alterations in blood flow and BP. Identification of the regulators of MYPT1 transcription and splicing will facilitate the study of the signaling pathways that mediate pressure/flow or hormonal-induced changes in MYPT1 expression in pregnancy, hypertension of pregnancy, and the other models that we have studied.

The shift to the MYPT1 LZ+ isoform in HtP was associated with increased sensitivity of the UAs to SNP-mediated relaxation, and an even larger leftward shift in the vasorelaxant dose-response to cGMP in permeabilized vessels with calcium clamped at submaximally activating concentrations. The ability of cGMP to induce relaxation in the presence of activating calcium, i.e., calcium desensitization of force production, as originally described by Kitazawa and coworkers (11; reviewed in Ref. 5), is the classic assay for cGMP-dependent activation of MP within the intact muscle. That the HtP UA is ~10-fold more sensitive to cGMP than the pregnant UA is consistent with the shift to the MYPT1 LZ+ isoform. The calcium-clamped cGMP dose-response of the HtP UAs is similar to that of the aorta (10), a tissue that expresses nearly 100% LZ+ MYPT1, and is consistent with the Km of the cGK1{alpha} for 8-Br-cGMP of 26 nM (23). The differences between the HtP, pregnant, and virgin UAs in the dose-response to SNP are eliminated with the phosphatase inhibitor tautomycetin, further supporting the premise that increased phosphatase activity is responsible for the increased sensitivity of the HtP UA to NO/cGMP signaling. Given that tautomycetin is not specific for MP, gene-based change-of-function studies are required to determine how much of this difference is specifically due to differences in MP activation.

One limitation of the present study is the absence of a direct indicator of MP activation in the different experimental groups. The regulation of MP activity is highly complex, which is not surprising given its critical role in regulating vascular tone and blood flow. There currently is no direct biochemical marker for MP activation. One report published while the present article was in revision suggests that cGK1 phosphorylation of MYPT1 at Ser695 activates MP and restores basal activity through the disinhibition of the {alpha}-adrenergic/Rho kinase-mediated inhibition of MP (17). It is not clear how this proposed mechanism would account for the ability of cGMP to activate MP and cause calcium desensitization in the absence of agonist stimulation as in the present and prior studies. Our direct measurements of MP activity suggest that the fraction of MP that is activated after SNP is greater in the HtP UAs as compared with virgin and pregnant UAs, consistent with the vascular relaxant responses to SNP and cGMP of these vessels. These measurements of MP activity were done in the absence of agonist preactivation, which could confound the measurements given that tissues may have differential sensitivity to agonist-induced MP inhibition possibly dependent on the expression of the MP inhibitor CPI-17 (27). Also confounding this issue of regulation of MP activity are 1) the expression of nearly equal ratios of MYPT1 LZ+:MYPT1 LZ– isoforms in pregnant and virgin UAs and 2) the approximately twofold higher level of expression of MYPT1 in pregnant UAs as compared with HtP and virgin UAs. It is well established that MYPT1 expression and MP activity is twofold to threefold higher in phasic as compared with tonic smooth muscle tissues (4, 19). The prototypical phasic tissues, e.g., rat portal vein and chicken gizzard, exclusively express the MYPT1 LZ– isoform, and these tissues fail to activate MP and desensitize to calcium after NO/cGMP activation (10, 19). These studies of intact muscle tissues support in vitro studies showing that the MYPT1 COOH-terminal LZ motif mediates heterodimerization with the cGK1 and is required for cGMP-dependent activation of MP (7, 25). It has yet to be determined how the expression of mixtures of MYPT1 LZ isoforms may influence vascular responses to NO/cGMP and other signaling pathways. It is interesting to note that the pregnant UA, which expresses approximately twofold more MYPT1 vs. HtP UA but at ~50:50 ratio of LZ+/– vs. HtP ~75:25 ratio, is ~10-fold less sensitive to cGMP-mediated calcium desensitization but achieves the same degree of maximal relaxation. One potential explanation is that the LZ– isoforms of MYPT1 are dominant over MYPT1 LZ+ in vivo at the myofilament, and this is overcome at higher concentrations of cGMP. This type of competition between MYPT1 isoforms for access to phosphorylated myosin would likely not be evident in measurements of MP activity in tissue homogenates in vitro. Alternatively, given the complexity of signaling pathways, it is possible that different molecules are involved in the cGMP-mediated calcium desensitization in HtP vs. pregnant UAs. In this regard, it is interesting to note that the Km of cGK1β for 8-Br-cGMP is ~10-fold higher than that for cGK1{alpha} (23), approximating the differences in the sensitivity of pregnant and HTP UAs to 8-Br-cGMP. cGK1{alpha}, but not cGK1β, is implicated in the activation of MP in MYPT1 LZ+ cells and tissues. The current study establishes a rationale for gain-, loss-, and change-of-function studies to define the role of these proteins in setting the sensitivity of the uterine circulation to NO/cGMP in pregnancy and hypertension of pregnancy.

In conclusion, we have shown MYPT1 switching to the LZ+ isoform specifically in the UA in a model of HtP. This is associated with increased sensitivity to the NO donor SNP and its second messenger cGMP and is prevented by chronic treatment with antihypertensive medicine and acute treatment with a type 1 phosphatase inhibitor. We propose that this isoform switch is an adaptive response to reduce vascular resistance and maintain uterine blood flow in the setting of hypertension-triggered inward remodeling of the UAs. Early and effective treatment of hypertension during pregnancy may be able to reverse some of the adverse effects on uterine perfusion and fetal development in this disease.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-66171 (to S. A. Fisher), HL-073895 (to G. Osol), and HL-073050 (to M. Ikebe).


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. A. Fisher, Dept. of Medicine (Cardiology), 4-533 Wolstein Research Bldg., 2103 Cornell Rd., Case Western Reserve School of Medicine, Cleveland, OH 44106-7290 (e-mail: saf9{at}po.cwru.edu)

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.


   REFERENCES
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 RESULTS
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