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Am J Physiol Heart Circ Physiol 280: H851-H858, 2001;
0363-6135/01 $5.00
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Vol. 280, Issue 2, H851-H858, February 2001

Reduced Na-K pump but increased Na-K-2Cl cotransporter in aorta of streptozotocin-induced diabetic rat

Luis Michea3, Verónica Irribarra1, I. Annelise Goecke2, and Elisa T. Marusic1

1 Laboratory of Cellular and Molecular Physiology, Faculty of Medicine, University of Los Andes, and 2 Institute of Biomedical Sciences, University of Chile, Casilla 20106, Las Condes 678-2468, Chile; and 3 National Institutes of Health, Bethesda, Maryland 20892-1603


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The activities of Na-K-ATPase and Na-K-2Cl cotransporter (NKCC1) were studied in the aorta, heart, and skeletal muscle of streptozotocin (STZ)-induced diabetic rats and control rats. In the aortic rings of STZ rats, the Na-K-ATPase-dependent 86Rb/K uptake was reduced to 60.0 ± 5.5% of the control value (P < 0.01). However, Na-K-ATPase activity in soleus skeletal muscle fibers of STZ rats and paired control rats was similar, showing that the reduction of Na-K-ATPase activity in aortas of STZ rats is tissue specific. To functionally distinguish the contributions of ouabain-resistant (alpha 1) and ouabain-sensitive (alpha 2 and alpha 3) isoforms to the Na-K-ATPase activity in aortic rings, we used either a high (10-3 M) or a low (10-5 M) ouabain concentration during 86Rb/K uptake. We found that the reduction in total Na-K-ATPase activity resulted from a dramatic decrement in ouabain-sensitive mediated 86Rb/K uptake (26.0 ± 3.9% of control, P < 0.01). Western blot analysis of membrane fractions from aortas of STZ rats demonstrated a significant reduction in protein levels of alpha 1- and alpha 2-catalytic isoforms (alpha 1 = 71.3 ± 9.8% of control values, P < 0.05; alpha 2 = 44.5 ± 11.3% of control, P < 0.01). In contrast, aortic rings from the STZ rats demonstrated an increase in NKCC1 activity (172.5 ± 9.5%, P < 0.01); however, in heart tissue no difference in NKCC1 activity was seen between control and diabetic animals. Transport studies of endothelium-denuded or intact aortic rings demonstrated that the endothelium stimulates both Na-K-ATPase and Na-K-2Cl dependent 86Rb/K uptake. The endothelium-dependent stimulation of Na-K-ATPase and Na-K-2Cl was not hampered by diabetes. We conclude that abnormal vascular vessel tone and function, reported in STZ-induced diabetic rats, may be related to ion transport abnormalities caused by changes in Na-K-ATPase and Na-K-2Cl activities.

vascular tone; endothelial modulation; hypertension


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

VASCULAR SMOOTH MUSCLE CELLS (VSMC) are major constituents of the blood vessel wall responsible for the maintenance of vascular tone. Na-K-ATPase maintains the Na+ electrochemical gradient across the VSMC plasma membrane and modulates contractility. Na-K-ATPase consists of two major subunits (alpha  and beta ) and a third small transmembrane protein, the gamma -subunit, that specifically associates with Na-K-ATPase in a tissue-specific manner (46). There are four alpha -catalytic subunit isoforms that show tissue-specific distributions. The existence of multiple catalytic isoforms, each with tissue-specific expression, suggests a specialized function.

alpha 1-, alpha 2-, and alpha 3-isoforms have been detected in rat vascular tissue and in cultured rat arterial myocytes (18, 31, 38). The localization of alpha 2-isoforms in microdomains of plasma membrane close to the sarco(endo)plasmic reticulum (18), as well as recent studies (17) in mice with genetically reduced levels of alpha 1- or alpha 2-isoforms, indicate a pivotal role for the alpha 2-isoform in VSMC in vivo in the control of intracellular Na+, calcium, and contractility. Epidemiological studies (37) in healthy subjects suggest that alpha 2-isoform contributes to the regulation of blood pressure.

The modulation of Na-K-ATPase activity and/or gene expression by insulin is of special interest for blood pressure regulation. Insulin decreases intracellular Ca2+ concentration in VSMC causing relaxation and reducing reactivity to vasoconstrictors (15, 19). The vasodilatory action of insulin has been shown to be at least partly mediated by Na-K-ATPase (43), probably because of cell membrane hyperpolarization (5, 42). Alterations in Na-K-ATPase activity and expression have been claimed to participate in hypertension and other diabetic complications (33). Studies in the streptozotocin (STZ)-induced diabetic rat model show highly tissue-specific changes in basal Na-K-ATPase activity, which decreases in cardiac muscle and neural tissue. Although only partially characterized, these changes correlate with abnormalities in alpha 1-, alpha 2-, and/or alpha 3-catalytic isoform expression (21, 32). A previous study shows decreased alpha 2-isoform mRNA levels in aortas from diabetic rats (34). However, the protein levels and/or functional consequences of the putative alpha 2 alteration in diabetic vascular tissue have not been reported.

Another relevant ion transporter of VSMC is the Na-K-2Cl cotransporter (NKCC1), which mediates the coupled electroneutral transport of 1 Na+, 1 K+, and 2 Cl- across the plasma membrane driven by the inwardly directed Na+ and Cl- gradients that occur under physiological conditions. NKCC1 is activated by cell shrinkage and is responsible for volume recovery, and it has been implicated in vascular tone and contractility as well as in hypertension. Bumetanide, a specific inhibitor of NKCC1 (7), inhibits the contraction of the rat aorta induced by norepinephrine (22), and vasoconstrictors or nitrovasodilators activate or inhibit NKCC1 in the rat aorta (1). ANG II increases NKCC1 in VSMC (40), and a significant increment in NKCC1 was found in hypertrophied VSMCs (48). Davis et al. (8) demonstrated that NKCC1 is involved in increased vascular Na+ permeability and membrane potential of deoxycorticosterone acetate-salt-induced hypertension, and we have shown an increase in the Na-K-2Cl activity in vascular tissue of hypertensive rats (12). Finally, recent studies (11) in the NKCC1-deficient mouse demonstrate that mean arterial pressure was significantly reduced in both heterozygous and homozygous animals, indicating an important function for NKCC1 in the maintenance of blood pressure. Because of the role of NKCC1 in cell volume regulation, it could also be activated in response to fluctuations in osmolality that can accompany diabetes. However, there are no reports of NKCC1 activity in diabetic vascular tissue.

Recent studies (6, 16) show that insulin-mediated vasodilation is endothelium dependent. Animal studies (30, 35) demonstrated impaired endothelium-dependent relaxation in aortas of both STZ-induced and genetically diabetic rats as well as normal rabbits exposed to hyperglycemia (44). Although nitric oxide (NO)-dependent and -independent pathways could be involved (5), the effectors of endothelial action on VSMC and its role in diabetes have not been established.

We hypothesize that alterations in sodium pump and/or NKCC1 in vascular tissue could increase vascular tone and contractility in vivo and participate in cardiovascular disease described in diabetic patients, i.e., hypertension (10, 20). In the present study, we investigated Na-K-ATPase catalytic isoforms and NKCC1 activity in the vascular tissue of control and STZ-induced diabetic rats. Furthermore, we studied the effects of the endothelium on Na-K-ATPase and NKCC1 activities in the aortic rings of control and diabetic rats.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Experimental Animals

Male Sprague-Dawley rats weighing between 175 and 190 g were used in this study. Diabetes was induced by a single STZ injection (65 mg/kg body wt ip). STZ was dissolved in 0.01 M citrate buffer; pH 4.5. Animals were confirmed to be diabetic by the presence of glucose in the urine and by blood glucose determinations. Treated and age-matched control rats were studied 4-6 wk after administration of STZ or vehicle. All of the animals had ad libitum access to standard rat chow and water throughout the experimental period. Blood samples were obtained from the tail vein for chemical determinations after 2 wk of STZ injection and on the experimental day for plasma creatinine and blood urea nitrogen.

Tissue Preparation and Incubation

Isolation of aortic rings. The rats were euthanized by decapitation. The thoracic aorta was quickly excised and placed in cold (4°C) physiological Krebs-Ringer bicarbonate (KRB) buffer containing (in mM) 4.2 KCl, 1.19 KH2PO4, 120 NaCl, 25 Na2HCO3, 1.2 MgSO4, 1.3 CaCl2, and 5 D-glucose (pH 7.4). Rings (4-6 mm and 2-4 mg/wt) were prepared after connective tissue was dissected from the aorta, taking special care to avoid endothelium damage. In some experiments, endothelium-denuded aortic rings were prepared by inserting a stainless steel wire into the lumen and gently rolling the ring on a filter paper soaked in KRB. At the beginning of the experiments, aortic rings from normal or diabetic animals were equilibrated for 1 h at 37°C in separate vials with 2 ml of KRB in a water-saturated atmosphere containing 95% O2-5% CO2 (Dubnoff incubator). After 60 min of incubation in KRB, tissue samples were used for transport experiments.

Isolation of skeletal muscle. Soleus skeletal muscles were isolated from control and diabetic animals. Groups of fibers weighing 8-15 mg were isolated and immediately incubated in separate vials with 2 ml of KRB for 60 min, as described for the aortic rings (4). After this period the fibers were used in transport experiments.

Isolation of cardiac muscle. Hearts were isolated from control and diabetic animals and gently rinsed with ice-cold KRB. Groups of fibers weighing 8-15 mg from the left ventricle were isolated and immediately incubated in separate vials with 2 ml of KRB for 60 min, as described previously (4). After this period, the fibers were used in transport experiments.

Sodium Pump Activity

Ouabain-sensitive 86Rb/K uptake was used as an index of Na-K-ATPase activity as described previously (4, 31). Briefly, the tissue samples (rings or muscle fibers in triplicate for each concentration) from control and diabetic rats were incubated in 2 ml of KRB in the absence or presence of ouabain (10-2 M to 10-7 M) for 15 min (aortic rings) or 30 min (muscle fibers). Thereafter, the tissue samples were transferred to vials containing 2 ml of incubation media (KRB supplemented with 0.1 µCi/ml of 86Rb) in the absence or presence of ouabain as indicated for 15 min. Transferring the samples into iced KRB stopped the reaction, and the tissues were then quickly washed in cold buffer and gently blotted. Sample radioactivity was determined by Cerenkov radiation after overnight solubilization with Triton X-100 (1%) in a liquid scintillation counter as described previously (4). Total pump activity was calculated by the difference between 0 and 10-2 M or 10-3 M ouabain; the high-affinity binding site, which refers to ouabain-sensitive activity, was measured at 10-5 M ouabain, and ouabain-resistant activity (alpha 1) was evaluated by the difference between total pump activity minus ouabain-sensitive activity (4, 31). The results are expressed as nanomoles 86Rb/K uptake per min per gram wet tissue.

NKCC1

Experiments studying the effect of diabetes on NKCC1 were performed identically to those indicated above for sodium pump activity except that, instead of ouabain, bumetanide was added at a final concentration of 50 µM to the preincubation and incubation media. NKCC1 activity was calculated by the difference between 86Rb/K uptake in the presence and absence of bumetanide 50 µM, as described by Bofill et al. (3).

Membrane Preparation and Western Blot Analysis

To minimize the potential selective enrichment of different pump isoforms during the purification procedure, we prepared a crude membrane fraction. The thoracic aorta free of adventitial tissue was washed in ice-cold KRB and processed immediately. Tissue from three animals was pooled for each preparation and was homogenized by a motor-driven Potter-Elvehjem Teflon homogenizer in ice-cold buffer containing 50 mM Tris · HCl, 1 mM phenylmethylsulfonyl fluoride, 2 µM leupeptin, 2 µM pepstatin, and 50 mM beta -mercaptoethanol; pH 7.4. The homogenate was centrifuged at 3,000 g for 10 min (4°C), and the supernatant was centrifuged at 100,000 g for 45 min (4°C). The membranes were suspended in 300 µl of 10 mM Tris · HCl, 10 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 10% glycerol (vol/vol), and 50 mM beta -mercaptoethanol, pH 7.4, and stored at -20°C.

SDS polyacrylamide gels were prepared according to the method of Laemmli (23). The blotting procedures were done according to Towbin et al. (47). After blotting was completed, the nylon membranes were blocked with 5% nonfat milk in Tris-buffered saline (Tris · HCl, 20 mM; NaCl, 137 mM) plus 0.1% Tween 20. After being washed once, membranes were incubated in the presence of mouse monoclonal specific anti-alpha 1 isoform of the Na-K-ATPase (provided by Dr. G. Kaplan, Yale University) and mouse monoclonal anti-alpha 2 McB2 (provided by Dr. Kathleen Sweadner, Harvard Medical School). The filters were subsequently washed and incubated with a secondary anti-mouse horseradish peroxidase-linked antibody (Amersham). The immune complexes were detected by an enhanced chemiluminescence method according to manufacturer's instructions (ECL; Amersham). Standard curves with increasing amounts of membrane homogenate (5-150 µg of total protein) were run to ensure linear response detection. Signals of each lane were quantified by computer scanning densitometry analyses, comparing the intensity of experimental and control rat samples as described previously (3, 4, 31).

Statistical Analysis

Values are expressed as means ± SE. For 86Rb/K inhibition experiments using various concentrations of ouabain, nonlinear least-squares analysis was performed with the use of the Nfit version 1.0 software package (University of Texas, Medical Branch, Galveston, TX). Densitometry volume data from Western blot experiments are expressed as percentage of the total volume intensity in the respective paired control sample. Statistical comparisons were accomplished by paired Student's t-test or by ANOVA (intact vs. denuded aortic rings). Difference was considered statistically significant if P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Characteristics of STZ-Induced Diabetic Rats

After 4-6 wk of STZ treatment, body weight was significantly reduced in the STZ-treated rats compared with control rats (Table 1). Plasma glucose levels of STZ-treated rats were significantly elevated after 2 wk (data not shown) and when the rats were euthanized (Table 1). There were no differences between the two groups in renal function, as measured by plasma creatinine and blood urea nitrogen (Table 1).

                              
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  		Table 1.   Effects of STZ-induced diabetes on body weight, plasma glucose, creatinine levels, and blood urea nitrogen

Effect of Diabetes on Na-K-ATPase Activity

We compared the total Na-K-ATPase activity in skeletal and vascular tissue of the diabetic rats and control animals. 86Rb/K uptake measurements were used as an index to evaluate the activity of Na-K-ATPase, calculated as the difference between 86Rb/K uptake without ouabain and 86Rb/K uptake in the presence of 10-2 M or 10-3 M ouabain. Preliminary experiments showed linear ouabain-sensitive 86Rb/K uptake for at least 20 min in these tissues (data not shown). As shown in Fig. 1, total sodium pump activity in aortic rings from diabetic rats decreased to one-half of the activity present in the vascular rings from control rats. However, in skeletal muscle, there was no significant difference between Na-K-ATPase activity of STZ rats compared with control animals.


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  		Fig. 1.   Effect of diabetes (DM) from streptozotocin (STZ) diabetic (D) rats on 86Rb/K uptake in skeletal muscle fibers and aortic rings. Skeletal muscle fibers and aortic rings without endothelium were incubated in Krebs-Ringer bicarbonate (KRB) buffer. Ouabain-sensitive 86Rb/K uptake was determined in the presence of 1 mM ouabain (see METHODS). Results are means ± SE of 7-9 experiments done in triplicate (no. of experiments in parentheses). C, control. *P < 0.01.

Functional Characterization of the Na-K-ATPase Isoforms

The catalytic isoforms of Na-K-ATPase in the rat differ in ouabain sensitivity; alpha 1-isoform is relatively ouabain resistant, whereas alpha 2- and alpha 3-isoforms are ouabain sensitive (27). Thus the contribution of ouabain-sensitive and ouabain-resistant isoforms to the total pump activity in vascular tissue can be distinguished by high ouabain (millimolar concentrations) or low ouabain concentrations (micromolar concentrations). The results of these experiments are summarized in Fig. 2, where aortic rings were incubated in KRB buffer containing ouabain concentrations ranging from 10-2 M to 10-7 M, as described in METHODS. The derived numerical values of the Na-K-ATPase activities associated with each ouabain-binding site and their respective inhibition constants (Ki) for control and diabetic tissues are included. A two-component inhibition model gave the best fit of the data (r2 = 0.999 for each curve). These data are similar to previous results from our laboratory in rat skeletal muscle and adipocytes (3) and to data from the literature (42). From ouabain inhibition data, we conclude that at ouabain concentrations in the range of 10-6 to 10-5 M, 90% of the ouabain-sensitive isoforms are inhibited, whereas <10% of the alpha 1-isoform is bound to ouabain (25). Ouabain (10-5 M) was then used to measure the ouabain-sensitive isoform activity, whereas 10-2 M ouabain was utilized for a complete inhibition of all alpha -isoforms. The summary of these analyses in Table 2 shows that the alpha 1-isoform is responsible for 56.3% of the total pump activity in the control aorta. The significant decrease in the total pump activity in the diabetic vascular tissue resulted from a dramatic decrease in ouabain-sensitive, 86Rb+-mediated uptake, which dropped to 18% of the total pump activity (26.1% of the normal level, P < 0.01).


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  		Fig. 2.   Ouabain inhibition of 86Rb/K uptake in aortic rings of C and DM rats. Aortic rings were incubated at 37°C for 30 min in KRB buffer containing concentrations of ouabain (see METHODS). Curves represent the best fit of the data to a two-component model, as determined by nonlinear least-squares analysis of 3 experiments done in triplicate. Ki, ouabain inhibition constant; M, area parameter of the respective component. K1 and M1, high-ouabain affinity component; K2 and M2, low-ouabain affinity component (r2 = 0.999).


                              
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  		Table 2.   Na+-K+-ATPase activity in aortic rings and relative contributions of ouabain-resistant and ouabain-sensitive catalytic isoforms

Western Blot Analysis to Determine Abundance of Na-K-ATPase Catalytic Isoforms

A decrease in alpha -isoform protein concentration in vascular tissue from diabetic rats can contribute to the decrease in Na-K-ATPase-mediated 86Rb/K uptake. We measured alpha 1- and alpha 2- isoform protein levels by Western blot analysis in crude membrane preparations isolated from the thoracic aorta (Fig. 3). In diabetic vascular tissue, both alpha 1- and alpha 2-protein levels were reduced; alpha 1-isoform was reduced to 71.3 ± 9.8% (P < 0.05) of control, and alpha 2-isoform was reduced to 44.5 ± 11.3% of control (P < 0.05).


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  		Fig. 3.   Rat aortic alpha -catalytic isoforms abundance in C and DM rats. Thoracic aortas were obtained after 28 days from 3 rats per group in each set of experiments. Crude membrane fractions were immediately processed, resolved by SDS-PAGE, and blotted as described in the text. A: representative immunoblots of aortic crude membrane fractions (100 µg) by using monoclonal anti-alpha 1- and anti-alpha 2-antibodies. B: bars represent means ± SE, 4 separate experiments, of immunoblot band densities for alpha 1 and alpha 2 abundance in C and DM rats. *P < 0.05 vs. C.

Role of Endothelium on Ouabain-Sensitive 86Rb/K Uptake

Previous work suggests that the endothelium affects Na-K-ATPase activity. We evaluated the effect of endothelium on Na-K-ATPase activity of control and diabetic rats by determining 86Rb/K uptake in aortic rings with intact endothelium compared with rings from which endothelium was previously removed. As shown in Fig. 4, intact or denuded diabetic aortic rings had significantly lower ouabain-sensitive 86Rb/K uptake compared with respective controls. Furthermore, from examination of the influence of endothelium on Na-K-ATPase activity, it may be concluded that the endothelium in diabetic tissue maintains its stimulatory action and stimulates a higher Na-K-ATPase activity compared with control tissue (130% increase in ouabain-sensitive 86Rb/K uptake in diabetic aorta vs. 50% increment in control aorta). Higher uptake values observed in the intact aortic rings versus denuded rings are not caused by 86Rb/K uptake by endothelial cells, because in control experiments we found that ablating the endothelium immediately after the uptake experiments (in ice-cold KRB) does not decrease the 86Rb counts compared with the parallel nondenuded control rings, in agreement with previously described results (12, 14).


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  		Fig. 4.   Endothelium-dependent stimulation of ouabain-sensitive 86Rb/K uptake. Aortic rings with and without endothelium were incubated with KRB, and 86Rb/K uptake was determined in the presence or absence of 10-2 ouabain to obtain the ouabain-sensitive component. Results are means ± SE of 7 separate experiments done in triplicate. E(+), aortic rings with endothelium; E(-), denuded rings. *P < 0.05 vs. C aortic rings with intact endothelium. **P < 0.01 vs. denuded C aortic rings and vs. intact D or C aortic rings.

Effect of Diabetes on NKCC1

Bumetanide, a specific inhibitor of NKCC1, was used to measure bumetanide-sensitive 86Rb/K uptake in aortic rings of control and diabetic rats. As shown in Fig. 5, bumetanide-sensitive 86Rb/K uptake increased significantly in diabetic aortic rings (160.5 ± 11.9 vs. 92.8 ± 10.5 nmol 86Rb/K · g wet wt-1 · min-1 in control rats). In contrast, the activity of the NKCC1 in ventricular muscles from control and diabetic rats was similar (63.8 ± 6.9 vs. 49.3 ± 5.3 nmol 86Rb/K · g wet wt-1 · min-1 in controls; n = 8 for each group). These results reemphasize the tissue specificity of diabetes on ion transport regulation.


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  		Fig. 5.   Bumetanide-sensitive 86Rb/K uptake in aortic rings with intact endothelium or without endothelium. Aortic rings of C and D rats were incubated in KRB with 50 µM bumetanide, where applicable. Bumetanide-sensitive 86Rb/K was calculated by subtracting uptake values obtained in the presence from uptake obtained in the absence of bumetanide. Results are means ± SE of 7-9 experiments, with each point assayed in triplicate. *P < 0.05, DE(-) vs. the other 3 conditions. **P < 0.01, CE(+) vs. CE(-) or vs. DE (+).

Role of Endothelium on Bumetanide-Sensitive 86Rb/K Uptake

Increased NKCC1 activity in diabetic arterial vessels was also observed in the absence of endothelium. As shown in Fig. 5, when the endothelium was removed, NKCC1 activity of diabetic vascular rings was four times greater than that of the control endothelium-denuded aorta. In diabetic animals, endothelium-denuded rings had ~70% of intact tissue activity, whereas activity dropped to 30% in denuded control aortas compared with intact aortic rings. These results indicate that bumetanide-sensitive cotransport in the diabetic aortic tissues is significantly increased regardless of the presence of endothelium. Also, these results suggest a modulating role of endothelium on NKCC1 activity. Previous studies (13) have shown that the higher values observed in intact aortic rings versus denuded rings are not caused by uptake in endothelial cells, because the contribution of the endothelium is minimal.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

A marked increase in the incidence of cardiovascular disease, e.g., hypertension, has been described in diabetic patients (10, 20). Increased peripheral vascular resistance appears to be a hallmark of hypertension in diabetic individuals. The etiology of hypertension in the majority of diabetic patients cannot be attributed to underlying renal disease and remains unexplained (9). Some evidence (1, 6, 35) suggests that diabetes may participate in the pathogenesis of hypertension through actions in smooth muscle tissue. Two studies (21, 32) have shown defective Na-K-ATPase activity in tissues such as the heart and the nervous system of diabetic animals. Altered Na-K-ATPase activities could result from altered enzyme kinetics and/or altered expression of their subunit proteins (24, 41). The present study examined whether STZ-induced diabetes, a model of insulin deficiency, alters the activity and the protein levels of the Na-K-ATPase catalytic isoforms in VSMCs. The results indicate that STZ-induced diabetes elicited isoform- and tissue-specific alterations in Na-K-ATPase activity in vascular tissue. In contrast, no functional alteration is present in the skeletal muscle from diabetic animals. The absence of abnormalities in skeletal muscle tissue is in agreement with data from previous studies (21, 32).

The defect in vascular tissue is because of a marked decrease in ouabain-sensitive activity in aortic rings. Takeshi-Ohara et al. (34) reported diminished total activity of the Na-K-ATPase in whole homogenates (NADH method) and lower mRNA levels for the alpha 2-isoform in the diabetic rat aorta. Our protein measurement data are consistent with their alpha 2-isoform mRNA data and additionally show a significant effect on alpha 1-isoform. Ng et al. (32) also demonstrated a marked decrease in alpha 2-protein levels in cardiac muscle of STZ-induced diabetic rats compared with control animals, and Vér et al. (49) showed that insulin treatment in STZ rats caused only a partial recovery of enzyme activity in heart tissue, although mRNA of catalytic isoforms were higher than in control rats.

The reduction in alpha 2-protein correlates with the reduction of the ouabain-sensitive component of the 86Rb/K uptake through the Na-K-ATPase. We recently found expression of alpha 1- and alpha 2-catalytic isoforms in the rat aorta, but we did not detect alpha 3-isoform mRNA (31). In addition, we did not detect alpha 3-protein in aortic membrane homogenates (Western blot) or in aortic sections (immunocytochemistry, see Ref. 31). However, others (18, 38) have detected alpha 3 expression in the rat tail artery and rat arterial myocytes in culture. Therefore, it is possible that alpha 3 is expressed in the rat aorta at levels under the detection limits of our previous experiments. If that is the case, alpha 2- and alpha 3-isoform activities are the functional components of the ouabain-sensitive 86Rb uptake. Thus the present data suggest that the reduction in alpha 2 expression in the aorta contributes to the decrease in the ouabain-sensitive 86Rb/K uptake observed in STZ-diabetic rats. It is also known that in traditional target tissues, insulin specifically increases the activity of the alpha 2-isoform, increasing its fraction of pumping (12) or its incorporation to plasma membrane (28). Therefore, in addition to reduced protein expression, it is possible that the lack of short-term insulin action in these animals could also contribute to selective reduction in alpha 2-isoform activity in vascular tissue in vivo.

A specific decrease in the alpha 2-isoform activity in VSMC could be functionally relevant in Ca2+ homeostasis and vascular contractility. A reticular distribution of the alpha 2-isoform has been demonstrated in the plasma membrane of arterial myocytes in culture, paralleling the endoplasmic reticulum (18). This has led to the proposal that alpha 2-isoform could regulate intracellular [Na+] in a restricted cytosolic space, affecting membrane potential and Ca2+ local concentration (18). Insulin has been shown to cause a decrease in intracellular Ca2+ levels in VSMC, causing relaxation and also reducing reactivity to vasoconstrictors (5, 19). This observation has been explained by the stimulatory action of insulin over Na/H+ exchanger and Na-K-ATPase activity in VSMC, leading to plasma membrane hyperpolarization and consequent closure of Ca2+ voltage-gated channels and activation of Na+/Ca2+ exchange (5). Interestingly, a recent study by James et al. (17) shows that hearts from mice with genetically reduced levels of alpha 2-isoform are hypercontractile as a result of increased calcium transients during the contractile cycle; in contrast, hearts from alpha 1-deficient mice are hypocontractile. Thus we speculate that the reduced expression and activity of the alpha 2-isoform in vascular tissue could be one of the direct effects of diabetes on VSMC, leading to increased and/or more sustained calcium transients, contributing to increased vascular tone and contractility.

An interesting observation of the present study is that diabetes caused not only a change in vascular sodium pump but also a dramatic modification in another important transporter, such as Na-K-2Cl. In the VSMC, the NKCC1 system is known to be regulated by endothelin, atrial natriuretic peptide, alpha - and beta -adrenergic agonists, and ANG II, which have a modulating role in vascular tone, suggesting that the activity of the NKCC1 may influence VSMC contraction and function (8, 48). Bumetanide inhibits the norepinephrine- and phenylephrine-induced contractions of the rat aorta (1, 22), and NKCC1 activity in the rat aorta is increased by vasoconstrictors and is reduced by NO and nitroprusside (1). On the other hand, the NKCC1-deficient mouse has decreased mean arterial pressure (11). Therefore, the current evidence indicates that the activity and further activation by vasoconstrictors of NKCC1 increases vascular tone and contractility. Our results indicate that NKCC1 activity was significantly increased in aortas of STZ rats (Fig. 5). In this context, changes in NKCC1 basal activity in vascular tissue can favor increases in vascular tone in diabetes. As previously proposed (1, 22), the increase in Cl- inward transport, elicited by increased NKCC1 activity, would lead to accumulation of intracellular chloride to a Cl- equilibrium potential value more positive than the membrane potential. Thus when chloride conductance is more dominant for the VSMC membrane potential, the NKCC1-mediated chloride accumulation would produce more Cl- efflux and depolarization. This would be the case when a vasoconstrictor binds to its receptor, increasing intracellular calcium and Cl- conductance. Considering that the acute activation of NKCC1 by vasoconstrictors in vascular tissue seems to be a direct consequence of intracellular Ca2+ increase (1), the coexistence of decreased sodium pump activity and increased NKCC1 activity could be two important interacting factors to increase VSMC contractility in diabetes.

No previous studies have evaluated NKCC1 activity in diabetic animals. It has been shown that insulin activates furosemide-sensitive K+ uptake in skeletal muscle-like cells (29) and in 3T3-LI adipocytes (39). Because of the proliferative phenotype in culture and because NKCC1 is activated by growth factors, studies in vascular tissue are relevant. Although the question of whether the NKCC1 activation plays an essential role in signal transduction and cell proliferation is under investigation, bumetanide and furosemide inhibited cell cycles in fibroblasts, and overexpression of NKCC1 induced proliferation and transformation (36). Thus it is conceivable that NKCC1 increased activity can favor VSMC proliferation in diabetic vascular tissue, or, alternatively, it could reflect abnormal smooth muscle proliferation associated with diabetes. It remains to be established whether or not the increment NKCC1 activity in the STZ rat aorta is a consequence of fluctuation in osmotic conditions and/or reflects more complex hormonal effects. Nevertheless, no significant changes were observed in heart tissue. The demonstration of similar effects of diabetes on NKCC1 activity in skeletal muscle and other insulin target tissues is relevant, because activation of Na-K-2Cl has been proposed as one of the putative mechanisms for rapid activation of Na-K-ATPase by insulin.

It has been demonstrated (16) that endothelial cells stimulate Na-K-ATPase activity in VSMC. Our results indicate that in the presence or absence of endothelium, diabetic rats have a diminished ouabain-sensitive 86Rb/K uptake compared with control tissue. These findings differ from data in the diabetic rabbit aorta, which showed a diminished ouabain-sensitive 86Rb uptake only when endothelium was present (14). These studies were carried out in alloxan-treated rabbits, and the differences observed may be because of differences in species, drug used, and/or the severity of the diabetes itself. Moreover, we find a significant stimulus by endothelium in the basal NKCC1. Nitrovasodilators inhibit the basal activity of NKCC1 and also reverse its activation by phenylephrine in the rat aorta (1). Nitroprusside is reported to reduce intracellular calcium in VSMC (26). All of these data, and the finding that endothelial cells are capable of producing numerous vasoactive substances, suggest that in the basal condition, a factor different from NO could be mediating the endothelial-stimulatory action over NKCC1 in the rat aorta. Future work should be directed to establish the factors involved in the NKCC1 stimulus in both normal and diabetic tissue.

The dissociation between reduced Na-K-ATPase activity and increased NKCC1 in STZ-induced diabetes differs from hypertensive rats, in which both systems are enhanced (8, 13). In addition to the opposite effects of vasodilators and vasoconstrictors on NKCC1 activity in rat aortas (1) and the findings in NKCC1-defficient mice (11), all of this evidence indicates an important role for NKCC1 activity influencing VSMC function. Our findings in the STZ rat aorta raise the possibility that one of the important consequences of diabetes in vascular tissue is a defect in these two transport systems, increasing vascular tone and/or contractility.


    ACKNOWLEDGEMENTS

We thank Dr. M. Caplan of Yale Medical School and Dr. K. Sweadner of Harvard Medical School for providing the anti-alpha 1- and anti-alpha 2-monoclonal antibodies and Rosario Flores and Javier Venegas for expert technical assistance. We also thank Dr. Joan D. Ferraris and Dr. Heddwen Brooks (Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute) for editing this manuscript.


    FOOTNOTES

This work was supported by grants from Fondo Nacional de Desarrollo Científico y Technológico 194/0524 and 197/0696 and Universidad de los Andes med 007.

Address for reprint requests and other correspondence: L. Michea, Facultad de Medicina, Universidad de los Andes, San Carlos de Apoquindo 2200, Las Condes 678-2468, Santiago, Chile.

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.

Received 1 May 2000; accepted in final form 19 September 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Heart Circ Physiol 280(2):H851-H858


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