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(Hypertension. 1996;27:740-745.)
© 1996 American Heart Association, Inc.

Articles

Increased Responsiveness and Decreased Expression of G Proteins in Deoxycorticosterone Hypertension

Nancy L. Kanagy; R. Clinton Webb

From the Department of Physiology, University of New Mexico School of Medicine, Albuquerque (N.L.K.); and the Department of Physiology, University of Michigan, Ann Arbor (R.C.W.).

Correspondence to Dr Nancy L. Kanagy, Department of Physiology, 237 Basic Medical Sciences Bldg, University of New Mexico School of Medicine, Albuquerque, NM 87131-5321.

	Abstract

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Abstract Deoxycorticosterone-salt (DOCA-salt) hypertension is characterized by elevated vasoconstriction to agonists that stimulate G protein–mediated activation of phospholipase C. However, the mechanisms responsible for the augmented responsiveness are unknown. This study tested the hypothesis that this augmented vascular responsiveness is due to elevated content of G_q, the G protein -subunit that activates phospholipase C. Thoracic aortae from DOCA-salt hypertensive rats (systolic blood pressure 183±7 mm Hg) and normotensive controls (systolic blood pressure 115±2 mm Hg) were homogenized and G protein content determined. Western analysis revealed that G_i content was decreased in DOCA compared with control rats (1364±196 versus 2343±188 densitometry units, P.05) with no differences observed for G_q or G_s. In addition, contractile responses in denuded femoral artery strips revealed a significant decrease in EC₅₀ values in DOCA arteries to all of the agonists examined: aluminum fluoride (DOCA=1.42, control=2.34 mmol/L), mastoparan (DOCA=0.51, control=35 µmol/L), phenylephrine (DOCA=0.08, control=0.53 µmol/L), and serotonin (DOCA=0.014, control=0.04 µmol/L, EC₂₀ values). Finally, arteries from DOCA rats contracted with aluminum fluoride had increased sensitivity to G protein antagonists but not to a phospholipase C inhibitor. The enhanced contractile responsiveness in the DOCA arteries may be mediated in part through decreased G_i levels. However, it is not caused by increased concentrations of G_q in the cell membrane or by increased phospholipase C sensitivity, and the increased constrictor response to G protein stimulators of phospholipase C appears to depend primarily on increased G protein sensitivity.

Key Words: aluminum fluoride • mineralocorticoids • G proteins

	Introduction

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Deoxycorticosterone-salt hypertension is characterized by increased contractility to multiple vasoconstrictors. The mechanism of this elevated sensitivity is partly dependent on increased transduction of the receptor-mediated signal to the contractile apparatus. Upregulation of signal transduction appears to be independent of changes in the receptor number or affinity¹ occurring at a postreceptor site. The event in signal transduction immediately after receptor stimulation is activation of a membrane-bound G protein that transmits the signal to an effector.² The G proteins that regulate vasoconstriction include the Gq family of -subunits that activate the ß-isoform of PLC (PLC_ß)³ ; the G_i family that inhibits adenylyl cyclase,⁴ inhibits calcium channels,⁵ and activates potassium channels⁶ ; and the G_s family that activates adenylyl cyclase⁷ and inhibits calcium channels.⁸ An increased responsiveness or quantity of G_q should lead to increased vasoconstriction dependent on increased PLC activation after receptor stimulation. Similarly, decreased activity or expression of G_s should lead to a decreased vasodilator response by decreasing the production of cAMP. The effect of decreased expression of G_i, however, is more difficult to predict. G_i activation has been associated with activation of PLC⁹ and vasoconstriction in some vascular beds, suggesting that inhibition of G_i-modulated pathways would lead to decreased vasoconstriction. Similarly, decreased G_i inhibition of adenylyl cyclase should lead to increased cAMP production and decreased vascular tone. However, G_i also has been linked to inhibition of calcium channels⁵ and activation of potassium channels,⁶ suggesting that decreased expression of G_i also could lead to depolarization and elevated calcium influx contributing to increased vasoconstriction.

Previous studies examining G protein content in tissues from DOCA rats found that G_i was increased in myocardial tissues.^{10 11} These studies also found decreased myocardial cAMP production, indicating that depressed adenylyl cyclase activity in failing hearts might be dependent on elevated levels of G_i protein. Studies in vascular tissue from hypertensive rats have produced mixed reports. Several models of hypertension have been shown to have increased G_i expression,¹² decreased G_i expression and elevated G_q expression,¹³ or unchanged levels of G protein subunit expression.¹⁴ Since there are conflicting reports in the literature regarding the relation between G-subunit expression and vascular contractility and no studies have examined vascular expression in this model of hypertension, the current investigation was conducted to determine whether altered expression of G proteins could contribute to increased vascular reactivity in DOCA-salt hypertension. We hypothesized that the elevated contractility in this model of hypertension is due to increased expression of G_q.

	Methods

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Drugs
Pertussis toxin was obtained from Research Biochemicals Institute, electrophoresis reagents were from BioRad, chemoluminescence reagents from Amersham Life Sciences, Inc, and antibodies from New England Nuclear. All other drugs and chemicals were obtained from Sigma Chemical Co. Stock solutions of drugs were prepared as follows and diluted in distilled water before being added directly to the muscle baths: (1) serotonin was dissolved in 0.1% ascorbic acid; (2) PE, AlF₄^-, sodium fluoride, sodium suramin, and benzalkonium chloride were dissolved in distilled water; and (3) NCDC and indomethacin were dissolved in ethanol. All drugs were added to the muscle baths in volumes not exceeding 0.50% of the total volume. Concentrations are expressed as molar values with respect to the final concentration in the tissue bath.

Animals
Adult male Sprague-Dawley rats (200 to 300 g; Harlan Animal Farms, Indianapolis, Ind) were used in this study. Rats were anesthetized with ether and uninephrectomized. Half of the rats received a subcutaneous silicone implant impregnated with DOCA (200 mg/kg). After surgery, DOCA rats were given 1% NaCl and 0.2% KCl in their drinking water. Control rats received no implant and drank tap water. Experiments were performed after 4 to 5 weeks of DOCA treatment. Systolic blood pressure was measured weekly in conscious rats using the tail-cuff method (pneumatic transducer). All animal protocols were approved by either the University of Michigan or the University of New Mexico committees for animal use. All protocols conformed to National Institutes of Health and university guidelines for the ethical treatment of animals.

Preparation of Tissues
On the day of the experiment, animals were anesthetized with sodium pentobarbital (60 mg/kg) and exsanguinated. Thoracic aorta and femoral arteries were removed and placed in physiological saline solution containing (mmol/L) NaCl 130, KCl 4.7, KH₂PO₄ 1.18, MgSO₄ · 7H₂O 1.17, NaHCO₃ 14.9, dextrose 5.5, CaNa₂ EDTA 0.026, and CaCl₂ 1.6. Femoral arteries were cleaned, cut into helical strips (1 mmx15 mm), and mounted in tissue baths filled with physiological saline solution maintained at 37°C and aerated with 95% O₂/5% CO₂ to achieve a pH of 7.2. All strips used were denuded of endothelium by gently rubbing the lumen of the strips with a cotton-tipped applicator. The removal of the endothelium was confirmed by failure of contracted strips (10^-7 mol/L PE) to relax to acetylcholine (10^-6 mol/L). Strips were stretched with a passive tension (700 mg) determined previously¹⁵ to allow maximal contraction in response to norepinephrine (5.9x10^-6 mol/L) and were equilibrated 2 hours before experiments were performed. Strips from a DOCA rat and a sham rat were paired in each bath and force-recorded with FT03 Grass Instruments transducers. Two strips from a single rat were used in antagonist studies, and no strip was used for more than two concentration-response curves. The number of animals used for each protocol is indicated by n values.

Experimental Protocols
Sensitivity to Contractile Agents
After equilibration, cumulative concentration-response curves were generated for two G protein stimulators: AlF₄^-, which binds to the GDP of the GTPase in the -subunit of G proteins to cause activation¹⁶ and mastoparan, a peptide that mimics a ligand-bound receptor to directly stimulate G protein activity.¹⁷ Cumulative concentration-response curves were also generated for PE, an ₁-adrenergic agonist, and serotonin. The concentration required to produce half maximal tension development (EC₅₀ value) was used to evaluate the sensitivity to contractile agents, while maximal tension development was used to evaluate the ability of the strip to develop tension and the efficacy of the contractile agent.

Sensitivity to Inhibitors
A second set of experiments evaluated the functional sensitivity of the G proteins causing AlF₄^- contraction by determining the sensitivity to G protein inhibitors in strips contracted with AlF₄^-. Cumulative concentration-response curves to inhibitors were obtained in strips contracted with AlF₄^- (6 mmol/L). Contractions are expressed as percentage of initial force. A third series of experiments evaluated the contribution of PLC stimulation to AlF₄^- contractions. PLC sensitivity was evaluated by constructing cumulative inhibitory concentration-response curves with the PLC inhibitor NCDC in strips contracted with AlF₄^- (6 mmol/L). NCDC has been demonstrated to inhibit PLC, and to a limited extent phospholipase A₂, in a concentration-dependent manner in intact cells.^{18 19} Elevated sensitivity at the level of the effector (PLC and/or phospholipase A₂) to G protein stimulation would be expected to cause increased sensitivity to the inhibitor, NCDC.

Western Analysis of G Proteins
Assessment of vascular smooth muscle G protein -subunit content was evaluated using a modification of the immunoblotting method described by Clark et al.¹⁴ Thoracic aortae were cleaned of connective and adventitial tissue, disrupted in a Dounce homogenizer containing ice-cold Tris-HCl buffer with EDTA (0.3 mg/mL) and phenylmethylsulfonyl fluoride (35 µg/mL), and the homogenate was spun at 800g for 10 minutes to remove cellular debris. The supernatant was drawn off and spun at 50 000g for 1 hour to isolate membranes. Membranes were resuspended in 500 µL buffer, an aliquot of each preparation was analyzed for protein concentration, and the remainder was stored at -70°C until used. Membranes were dissolved in sample buffer (1 µg/µL), boiled for 3 minutes, and separated on 12% polyacrylamide gels. In addition to paired DOCA and sham samples (25 µg/lane), each gel contained molecular-weight standards and purified G protein standards. Separated proteins were transferred to membranes and probed with monoclonal antibodies specific for the different G protein -subunits; AS/7 recognizes both i1 and i2 isoforms, QL is specific for q, while RM/1 recognizes Gs. Gels were stained with Coomassie blue to evaluate sample loading, and enhanced chemoluminescence development was used to visualize proteins. The relative quantity of protein was estimated using densitometry and molecular-weight markers and purified G proteins were used to verify antibody specificity.

After immunoblotting for G protein -subunits, some blots were stripped and reprobed with an antibody to ß-actin (Sigma). These blots were used to evaluate smooth muscle content in each group.

Data Analysis and Statistics
Data are reported as the mean±SEM. For calculation of EC₅₀ values (concentration that caused 50% maximal response), a logit-log transformation was performed and the transformed data were curve-fitted by using an unweighted least-squares linear regression. Since not all agents caused maximal responses, threshold values were calculated using a one-way ANOVA with the least significant difference post-hoc test. The lowest value to cause a significant difference from control is designated the threshold. Unpaired Student's t tests were used to compare systolic blood pressures, absolute force measurements, and EC₅₀ values of the transformed data between animal groups. When multiple t tests were used for comparisons between groups, the Bonferroni adjustment for multiple testing was employed. A value of P<.05 was considered statistically significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Hypertension developed in all rats during the 4-week treatment period (Table 1). Rats were matched by age, and hypertensive rats had a significantly lower body weight by the end of the treatment period. Results of the specific experimental protocols are outlined below.

View this table: [in this window] [in a new window]   Table 1. Systolic Blood Pressure and Body Weight of Rats Throughout the Treatment Period

Sensitivity to Contractile Agents
Femoral arteries from DOCA rats had a significant increase in sensitivity to both AlF₄^- and mastoparan, apparent as decreased EC₅₀ values and leftward shifts in the concentration-response curves (Table 2 and Fig 1). In addition, the time to half-maximal tension development for an EC₈₀ concentration of AlF₄^- was significantly less in DOCA tissues, while total tension developed was greater in artery segments from control rats (Fig 2). DOCA tissues were also more sensitive to PE and serotonin, especially at low concentrations (Fig 3). These results indicate that the contractile response to G protein stimulation in arteries from DOCA-hypertensive rats is upregulated through a pathway at least partially independent of receptor stimulation. Similar shifts in AlF₄^- and PE concentration-response curves were seen in three experiments conducted in aortic ring segments (data not shown). These experiments indicated that the functional response to these agents is qualitatively similar between the two vascular preparations.

View this table: [in this window] [in a new window]   Table 2. EC50 Values in Femoral Arteries From Normotensive Sham Rats and Hypertensive DOCA Rats

View larger version (16K): [in this window] [in a new window]   Figure 1. Cumulative concentration-response curves to G protein stimulators in femoral arteries from DOCA hypertensive rats and normotensive sham rats. Left, Arteries were contracted with increasing concentrations of mastoparan. Force is expressed as a percent of the maximal contraction to KCl (90 mmol/L). Right, Sodium fluoride was added in increasing concentrations to arterial segments in the presence of aluminum chloride (20 µmol/L). Force is expressed as percent of the maximal tension developed. For each panel, points represent the mean of the number of animals indicated in parentheses, and the error bars are the SEM. *Significant differences from sham tissues for P.05.

View larger version (27K): [in this window] [in a new window]   Figure 2. Time course of AlF4- contraction (6 mmol/L). Tension development was recorded over a 30-minute period in femoral arteries from six DOCA and six sham rats. Points represent the mean (number of animals indicated in parentheses), and the error bars are the SEM. *Significant differences from sham tissues for P.05.

View larger version (14K): [in this window] [in a new window]   Figure 3. Cumulative concentration-response curves to PE and serotonin in femoral arteries from DOCA and sham rats with the number of animals indicated in parentheses. , Data from the DOCA tissues; , data from the sham tissues. *Significant differences between groups for P.05.

Sensitivity to Inhibitors
AlF₄^- contractions were reversed by both sodium suramin and benzalkonium chloride (Fig 4). Tissues from DOCA rats were more sensitive to both G protein antagonists evidenced by the lower threshold for relaxation (Fig 4). In contrast, both groups of tissues exhibited equivalent sensitivities to the PLC inhibitor, NCDC (Fig 5). This indicates that the increased sensitivity to G protein stimulation is not dependent on increased responsiveness of PLC. Rather, the upregulation in the G protein pathway appears to depend on increased sensitivity of the G proteins themselves.

View larger version (33K): [in this window] [in a new window]   Figure 4. Concentration-response curves to G protein inhibitors in tissues contracted with AlF4- (6 mmol/L). Points represent the mean of six rats, and error bars indicate SEM. Tissues were contracted with AlF4- (6 mmol/L) to generate control values, then treated with increasing concentrations of the agonists. The insets list the EC20 and EC50 values and the initial force developed before the addition of the antagonists. *Significant differences from responses in sham tissues; significant differences from control values within a group. The number of animals in each group is indicated in parentheses.

View larger version (31K): [in this window] [in a new window]   Figure 5. Concentration-response curves to PLC inhibitor NCDC in tissues contracted with AlF4- (6 mmol/L). , Data from DOCA rats; , data from control rats. *Significant differences between sham and DOCA tissues for P.05; significant differences from control values within a group. The numbers in parentheses indicate the number of animals for each group.

Western Analysis of G Proteins
Western analysis of vascular tissue revealed a significant decrease in the expression of Gi -subunits but no changes in Gs or Gq expression (Fig 6). A single band was observed in blots probed for G_i, indicating that only a single isoform was present. Since the antibody recognizes both i1 and i2 isoforms but only i2 is present in vascular tissue,²⁰ it appears that the change was in i-2 expression. When blots were analyzed for ß-actin, there was no difference in the amount of total protein loaded per well so that the decreased expression appears to be independent of changes in vascular smooth muscle mass (Table 3).

View larger version (47K): [in this window] [in a new window]   Figure 6. Changes in G protein -subunit expression in aortic tissue from DOCA. The inset shows scans of immunoblots for each of the three subtypes of G proteins examined for aortic samples from DOCA (D) and sham (S) rats and for a purified G protein standard (G). The antibody to Gi, AS/7, recognized one band of 41 kD in the membranes confirmed by binding to purified Gi1 protein. The antibody to Gs, RM/1, revealed bands at both 42 and 45 kD, confirmed by binding to purified Gs (45-kD form). The antibody to Gq, QL, recognized a 42-kD band, confirmed by binding to a crude lysate of Sf9 cells overexpressing Gq. The bar graph depicts densitometry readings of scanned blots for each of the three antibodies. The only significant difference was a decrease in the amount of Gi present in DOCA tissues.

View this table: [in this window] [in a new window]   Table 3. Densitometry Values of the Area Under the Curve of Scanned Western Blots

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Vascular contractility is under the control of multiple modulators. However, it is apparent that changes in the signal transduction pathways regulated by heterotrimeric GTP-binding proteins could contribute to the elevated sensitivity to vasoconstrictors.²¹ The current study investigated changes in vascular contractility and changes in vascular expression of G protein -subunits during DOCA-salt hypertension. There were two primary findings. First, an increased sensitivity to contractile agents acting through G protein–regulated pathways was observed. However, the increased sensitivity was not accompanied by increased expression of G_q as predicted, and it appears that the elevated response is not caused by an increased number of G_q proteins in the membrane. Second, there was a significant decrease in the content of Gi -subunits, and this may have contributed to the elevated contractility as discussed in the beginning of this article.

Vascular contractility to receptor-mediated agonists in the DOCA-salt model of hypertension is consistently found to be increased.^{15 22} This model of hypertension is induced by giving high doses of the mineralocorticoid deoxycorticosterone in combination with elevated salt intake. Within 1 week, blood pressure is elevated and remains elevated for the duration of high salt intake.²³ Initially, the hypertension appears to depend on sodium and water retention at the kidney, but the sustained phase of the hypertension is maintained by increased vascular resistance accompanied by elevated sympathetic outflow²⁴ and increased vasoconstrictor responses to endogenous stimuli.²² The current study investigated the mechanisms responsible for the elevated vasoconstrictor responses to receptor-mediated agonists in the established phase of hypertension.

It has previously been shown that during this phase of DOCA hypertension, vascular smooth muscle does not exhibit increased receptor number but does have an increased calcium signal in response to receptor stimulation.^{15 25 26} Therefore, changes in contractile sensitivity appear to be mediated by altered signal transduction at a point distal to receptor stimulation. In addition, contractile proteins in vascular tissue from DOCA rats do not have increased sensitivity to calcium,²⁶ further indicating that the changes in vascular responsiveness are dependent on altered signal transduction. However, it has been difficult to directly relate altered expression or function of signal transduction components to the increased vascular reactivity.

Previous reports in other models of hypertension have examined the vascular smooth muscle expression level of the different G protein -subunits that regulate contraction in smooth muscle cells: _i, _q, and _s. In genetic models of hypertension, myocardial content of G_i has been reported to be increased.¹² However, in vascular tissue from spontaneously hypertensive rats, levels of G_i and G_q appear to be unchanged,¹⁴ while in at least two experimental models of hypertension, G_i has been shown to be decreased and G_q to be increased.¹³ The expression of G-subunits in vascular tissue from DOCA rats has not been reported previously, although at least two groups have reported an increase in G_i expression in myocardial tissue.^{10 11} Therefore, it was hypothesized that arteries from DOCA rats would have increased expression of G_q, the G protein subtype commonly associated with PLC activation, as well as an increase in G_i, the subtype associated with inhibition of adenylyl cyclase and calcium channels and stimulation of potassium channels. It was expected that the altered G protein expression would be accompanied by increased sensitivity to G protein inhibitors but not to PLC inactivation and by increased sensitivity to contractile agents that activate G proteins (ie, AlF₄^-). However, Western analysis found a significant decrease in the amount of immunoreactive G_i and no apparent change in the expression of G_q or G_s. This was accompanied by a dramatic increase in the vasoconstrictor response to G protein stimulation with AlF₄^- and mastoparan. Because sodium fluoride inhibits phosphatase activity at millimolar concentrations,²⁷ the contractions induced by the combination of Al⁺³ and sodium fluoride in this study may be partially dependent on phosphatase inhibition. However, the highest concentration of AlF₄^- used, 12 mmol, causes only 50% inhibition of phosphatase activity in an isolated system,²⁸ and it is unlikely that phosphatase inhibition could account for the entire contraction produced in the vascular strips. In addition, the phosphatase inhibition induced by AlF₄^- may be a downstream event of the activation of a G protein²⁹ that also contributes to the contractile response of G protein–coupled receptors in vascular smooth muscle. This pathway is an important area for future studies in vascular contractility.

The reason for the differences between the observed results and the hypothesis is not immediately clear, but it is apparent that an increase in G_q is not responsible for the elevated contractile response to G protein stimulators. Rather, a decrease in Gi -subunits may contribute to the elevated vascular reactivity as described below.

A previous investigation also reported decreased G_i-2 expression in aortic tissue from experimentally hypertensive rats,¹³ although myocardial tissue from DOCA rats appears to have elevated G_i expression accompanied by decreased adenylyl cyclase production.^{10 11} The differences are most likely due to the different tissues examined. Importantly, the differences observed between models of hypertension indicate that blood pressure alone does not regulate aortic expression of G protein -subunits. The antibody used in the current study recognizes transducin, G_i-1, and G_i-2, but only G_i-2 is appreciably expressed in vascular smooth muscle.²⁰ Therefore, our data indicate that G_i-2, the subtype linked to adenylyl cyclase inhibition,⁴ K⁺ channel activation,⁶ and Ca²⁺ channel inhibition,³⁰ is decreased in DOCA-salt vasculature, a change that would decrease rather than increase vascular reactivity. Therefore, decreased G_i mediation of K⁺ channel activation and Ca²⁺ channel inhibition is more likely to contribute to the contractility changes observed. However, the small changes in Gi expression would not be expected to fully account for the augmented vascular response to G protein stimulation.

In summary, the current study found increased sensitivity of vascular tissues to G protein activation that was not accompanied by changes in Gq protein content. Therefore, the characteristic increase in PLC activation in DOCA hypertension³⁰ might depend instead on changes in the activation sensitivity of the G proteins. It appears that G protein function is regulated separately from expression levels and that regulation of G protein function can alter vascular reactivity.

	Selected Abbreviations and Acronyms

 

AlF4- = aluminum fluoride DOCA = deoxycorticosterone-treated rats NCDC = 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate PE = phenylephrine PLC = phospholipase C

	Acknowledgments

 
This work was supported by NIH grants HL-18575 and HL-09099 and New Mexico Heart Association grant NMBG-03-95.

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