Skip Navigation

Cardiovascular Research 1997 34(1):137-144; doi:10.1016/S0008-6363(96)00238-6
© 1997 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Smith, J. M
Right arrow Articles by M. Solar, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Smith, J. M
Right arrow Articles by M. Solar, S.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 1997, European Society of Cardiology

Na+/K+-ATPase activity in vascular smooth muscle from streptozotocin diabetic rat

Jacquelyn M Smith*, Dennis J Paulson and Suzanne M. Solar

Department of Physiology, Midwestern University, 555 31st Street, Downers Grove, IL 60515, USA

* Corresponding author. Tel. +1 708 515-6390; Fax +1 708 971-6414.

Received 25 March 1996; accepted 6 June 1996


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objectives: Insulin-deficient diabetes impairs carbohydrate metabolism in a variety of tissues. Vascular smooth muscle may be susceptible to the diabetes-induced disturbance in glycolysis since Na+/K+-ATPase in this tissue preferentially utilizes ATP generated by glycolysis. The purpose of this study was to determine if chronic exposure to the metabolic alterations associated with insulin-deficient diabetes directly inhibited Na+/K+-ATPase activity, or its regulation, in vascular smooth muscle. Methods: Diabetes was induced by intravenous administration of streptozotocin (60 mg/kg). After 12 weeks, Na+/K+-ATPase activity in aorta and superior mesenteric artery was evaluated under a variety of conditions. Na+/K+-ATPase was estimated by measuring the influx of rubidium-86 (86Rb) in the presence or absence of the Na+/K+-ATPase inhibitor, ouabain. The metabolism of [3H]glucose and [14C]glucose was used to estimate glycolysis or glucose oxidation, respectively. Results: Glycolysis and glucose oxidation were decreased in aortic smooth muscle (27 and 34%, respectively). An intact endothelium was associated with a marked decrease in ouabain-sensitive (pump-mediated) 86Rb uptake in diabetic aorta. However, ouabain-sensitive 86Rb uptake was similar in de-endothelialized aorta and superior mesenteric artery from diabetic and non-diabetic rats under both unstimulated conditions and during maximal stimulation. Removal of glucose or oxygen reduced ouabain-sensitive 86Rb uptake to a similar extent in both groups. In contrast, the receptor-mediated stimulation of ouabain-sensitive 86Rb uptake by insulin was decreased. Conclusions: These results suggest that intrinsic Na+/K+-ATPase activity is not diminished in diabetic vascular smooth muscle under physiological conditions and that the impairment of cellular metabolism in diabetic blood vessels does not limit stimulation of Na+/K+-ATPase activity. However, modulation of Na+/K+-ATPase activity by endothelial factors or insulin appears to be altered in aorta from diabetic rats.

KEYWORDS Na+/K+ ATPase; Na+/K+ ATPase inhibitor; Rat, aorta; Rat, arteries; Glycolysis; Glucose oxidation; Diabetes


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Diabetes has profound effects on the metabolism of a variety of tissues including vascular smooth muscle [1]. In blood vessels from diabetic animals, both glycolysis and glucose oxidation are decreased [2]and there is an overall decrease in ATP production [3]. One mechanism that may contribute to this disturbance in tissue metabolism is the elevation in long-chain fatty acids [4]or glucose which alters substrate utilization in diabetic tissues [5, 6]. For example, the excessive oxidation of lipids in insulin-deficient tissues has been shown to inhibit crucial enzymes in the glycolytic pathway including pyruvate dehydrogenase, phosphofructo-1-kinase and hexokinase [5, 7].

Transmembrane ion movement in vascular smooth muscle may be particularly susceptible to the inhibition of glycolytic enzymes associated with diabetes. Vascular smooth muscle is unusual in that aerobic glycolysis accounts for 15–30% of the basal ATP production [8–10]. Subsequent evaluation of the role that aerobic glycolysis may play in vascular smooth muscle demonstrated a functional correlation between aerobic glycolysis and Na+/K+-ATPase activity. Thus, aerobic glycolysis was increased when Na+/K+-ATPase was increased and decreased when Na+/K+-ATPase activity was decreased [10, 11]. These observations led to the proposal that Na+/K+-ATPase in vascular smooth muscle preferentially utilizes the ATP generated by glycolysis rather than oxidative metabolism, a selectivity attributed to the compartmentalization of the glycolytic enzymes in the plasma membrane [9, 11, 12]. In normal blood vessels, however, the dependence of the Na+/K+-ATPase on ATP generated via glycolysis is not unconditional. Oxidative metabolism may serve as an alternative source of ATP for Na+/K+-ATPase and maintain normal ion gradients in the absence of exogenous glucose [10, 13]. However, it is feasible that the impairment of cellular metabolism in diabetic blood vessels may limit this shunt to oxidative phosphorylation which, in turn, could decrease Na+/K+-ATPase activity.

There have been reports that the Na+/K+-ATPase activity was diminished in blood vessels from insulin-deficient diabetic rats [14, 15]. However, recent studies have demonstrated that endothelium itself releases chemical messengers that modulate the Na+/K+-ATPase activity in vascular smooth muscle [14]. So it is not clear whether Na+/K+-ATPase activity was diminished as a result of the impaired cellular metabolism in diabetic vascular smooth muscle itself, or reflected a change in the release of chemical messengers from the endothelium. The purpose of this study was to determine if chronic exposure to the metabolic alterations associated with insulin-deficient diabetes inherently altered Na+/K+-ATPase activity or its regulation in vascular smooth muscle. Therefore, Na+/K+-ATPase activity in aorta and superior mesenteric artery from streptozotocin-diabetic rat was evaluated in the absence of endothelium under a variety of conditions which included sodium-loading in combination with varying extracellular potassium concentrations, the absence of glucose or oxygen, and in the presence of insulin.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
Male Sprague-Dawley rats (150–200 g, Sasco Animal Laboratories, Madison, WI) were given an intravenous injection of streptozotocin (60 mg/kg) dissolved in freshly-prepared 0.1 M citrate buffer (pH 4.5). The animals were then maintained on normal rat chow and H2O ad libitum. After 12 weeks, body weight (g) was 341±9 in the diabetic animals and 599±13 in the controls. Diabetic animals exhibited elevated plasma levels of glucose, glycosylated hemoglobin, triglycerides, free fatty acids and cholesterol. These values are presented in Table 1 and are similar to those reported by Paulson et al. [4]. The animals were anesthetized with ketamine (80 mg/kg) and xylazine (8 mg/kg) i.p. The thoracic aorta and/or superior mesenteric artery (SMA) were removed, placed in dissection solution and cleaned of fat and connective tissue. The blood vessels were slit lengthwise and the endothelial cells removed by lightly stroking the intimal surface with moistened filter paper. Preliminary experiments determined that this procedure eliminated the endothelial-mediated vasodilation of pre-contracted aorta by acetylcholine. In one series of experiments, Na+/K+-ATPase activity was evaluated in the presence of an intact endothelium. The blood vessels were cut in half and each half was mounted on a stainless steel holder. Although the aorta is sparsely innervated [16], the superior mesenteric arteries required denervation [17]. Briefly, the two halves of the superior mesenteric artery were incubated for 10 min in modified Krebs' solution (see below) containing 300 µg/ml 6-hydroxydopamine, 1 µM phentolamine and 130 µM glutathione and bubbled with nitrogen. After 10 min, the tissue was rinsed in a similar solution but without 6-hydroxydopamine. Preliminary experiments determined that the denervation procedure itself did not alter Na+/K+-ATPase activity in the mesenteric artery.


View this table:
[in this window]
[in a new window]

  		Table 1 Blood glycosylated hemoglobin (gHb), plasma glucose, trigylcerides (TG), total cholesterol (Chol), HDL-cholesterol (HDL-Chol) and free fatty acids (FFA) in control (n=14) and diabetic (n=15) male Sprague-Dawley rats

 
Aorta or denervated superior mesenteric arteries were placed in physiological salt solution (PSS) or potassium-free PSS (sodium-loaded tissues) for 3 h at 37°C and bubbled with 97% O2/3% CO2. In selected experiments, glucose or oxygen was absent during the equilibration period. In the latter case, the tissues were bubbled with 97% N2/3% CO2. In the relevant protocol, insulin (Humulin U, Eli Lilly and Company, Indianapolis, IN) was added to the PSS during the last hour of the equilibration period.

Additional experiments evaluated the effect of weight reduction and insulin treatment on Na+/K+-ATPase activity. To test the effects of weight reduction, the food intake of control rats was restricted to equal 75% of the diabetic food intake. At this degree of food restriction, the weight gain of the control non-diabetic rats was comparable to that of the streptozotocin-diabetic rats over the 12-week period. In separate experiments, streptozotocin-diabetic rats were injected with 1 U/100 g body weight/day of Ultralente Iletin I insulin (Eli Lilly and Co., Indianapolis, IN). Insulin injections were started 2 weeks after the rat had received the streptozotocin and continued for 10 weeks.

2.1 Na+/K+-ATPase activity
Na+/K+-ATPase was estimated by measuring the influx of 86Rb which can be used as a substitute for K+ [18]. At the end of the equilibration period, one half of each blood vessel was placed in equilibration solution to which 86Rb (20 µCi/ml) had been added. The other half of each blood vessel (pre-incubated with 2 mM ouabain for 1 min) was placed in an identical radioactive equilibration solution +2 mM ouabain for 10 min. Ten minutes was the period chosen for influx measurements since rubidium uptake is linear during this period and there is minimal backflux [13, 19]. When the potassium concentration was altered in the uptake solution, the amount of 86Rb added to the uptake solution was adjusted to maintain a constant specific activity. Phentolamine, 1 µM, was added to all influx solutions [18]. At the end of this uptake period, the tissues were passed through a series of efflux tubes containing non-radioactive potassium-free PSS at room temperature (21°C). Radioactivity in the efflux tubes and the tissue was measured by liquid scintillation. Washout curves were computed (IBM-PC) by sequentially adding tissue and tube radioactivity in reverse order and normalized in terms of initial activity. Uptake of 86Rb by the tissue at 10 min was determined by log-linear analysis of the efflux curves and extrapolated back to zero time. Active 86Rb uptake was defined as ouabain-sensitive 86Rb uptake and calculated by subtracting the 86Rb uptake measured in the presence of 2 mM ouabain (ouabain-insensitive 86Rb uptake) from the total 86Rb uptake measured in the absence of ouabain. Since the uptake period was 10 min and the [K]o during uptake did not exceed 10 mM 19, the effects of backflux and hyperpolarization on net rubidium uptake were minimized and therefore no correction factors were used in the calculation. The cellular pool of 86Rb was estimated [18]and determined to be similar in aorta from control (135±4 µM/g dry weight) and diabetic (130±5 µM/g dry weight) animals (n=4). Therefore, Na+/K+-ATPase activity was expressed as the uptake of 86Rb/g dry weight of tissue/min.

2.2 Measurement of glycolysis and glucose oxidation
Adventitia was removed from aorta used in the metabolic experiments to facilitate the diffusion of substrates and oxygen during the 2-h incubation of the vessel in a closed system. To facilitate removal of the adventitia, each aorta was incubated at 37°C in PSS+Hepes (10 mM) containing collagenase (1 mg/ml; Worthington Biochemical Corporation, 300 U/mg) and trypsin inhibitor (1 mg/ml; Sigma). After 15 min, the aorta was rinsed in dissection solution and the adventitia removed. The remaining intima-media component of each aorta was placed in 5.5 ml of PSS fatty-acid-free bovine serum albumin (3 g/100 ml) containing [5-3H]glucose and [U-14C]glucose that had been previously gassed for 20 min with 97%O2/3%CO2. Preliminary experiments had determined that the rate of glycolysis and glucose oxidation was constant between 1 and 3 h of the incubation period. Therefore, the tissue was incubated in a closed system for 2 h. 5-3H2O, released at the enolase step of glycolysis, was used to measure steady-state glycolysis rates. 14CO2, released at the pyruvate dehydrogenase step and in the tricarboxylic acid cycle, was used to measure glucose oxidation rates [7]. Aliquots were withdrawn from the incubation medium each hour. Each aliquot was immediately injected below 1 ml of mineral oil and frozen at –70°C until analyzed. These perfusate samples were designated for measurement of glycolysis using the method described by Kobayashi and Neely [20]. After 2 h of incubation, 300 µl of 1 M hyamine hydroxide, a CO2-trapping agent, was added to the center well in the metabolic flask. H2SO4 (0.5 ml of 9 N) was then added to the medium in the flask to release dissolved CO2 and the incubation continued for an additional hour. At the end of this period, the hyamine hydroxide in the center well was placed in a vial to which 4 ml of scintillation fluid (Ultima Gold; Packard) was added. The samples were then counted on a beta scintillation counter (Tricarb 1900; Packard). The total CO2 generated by oxidation of glucose was determined from the specific activity of the incubation medium. The data are presented as µmol glucose oxidized/g dry weight/h.

To measure glycolysis, 3H2O was separated from [3H]glucose and [14C]glucose using columns containing Dowex 2-X8 anion exchange resin (200–400 mesh) suspended in 0.4 M potassium tetraborate [20]. The Dowex columns retain >99% of the [3H]glucose and [14C]glucose and the 3H2O is washed through the column. A 0.1 ml aliquot of each sample from the incubation medium was added per column (run in triplicate) and the 3H2O eluted from the column into scintillation vials with a 1.5 ml H2O wash. Scintillation fluid was added to each vial and the samples were counted with standard double isotope counting procedures using the beta-scintillation counter. The 3H2O counted in the eluent was corrected for the small amount of [3H]glucose (<1%) that passed through the columns and the spillover of 14C into the 3H window. The rate of glycolysis for each aorta was determined using the specific activity of the incubation and the data are presented as µmol glucose utilized/g dry weight/h.

2.3 Solutions
The normal physiological salt solution (PSS) had the following composition (in mM): Na+, 146.2; K+, 5.0; Mg2+, 1.2; Ca2+, 1.5; Cl–, 143,9; HCO3–, 13.5; H2PO4, 1.2; glucose, 11 mM. At 11 mM glucose, glucose utilization by the tissue is not limited by glucose transport [21, 22]. Hepes (10 mM) was included in the PSS used during the incubation with collagenase/trypsin inhibitor. The dissection solution was similar to PSS except that the calcium concentration was decreased to 0.25 mM. In experiments in which the tissues were equilibrated in potassium-free PSS, potassium was also removed from the dissection solution to facilitate sodium-loading the tissues. Modified PSS used in the denervation procedure did not include K+, NaH2PO4 and NaHCO3.

2.4 Statistics
Data are presented as mean±s.e.m. Analysis of variance followed by Fisher's least significant difference multiple-comparison test was used in the determination of statistical difference between 3 or more groups. The unpaired Student t-test was used to determine statistical significance between two groups. A P-value of <0.05 was considered significant.

This investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985).


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Glycolysis and glucose oxidation in diabetic aorta
Measurement of glucose utilization confirmed that both aerobic glycolysis and glucose oxidation were markedly decreased in aorta from streptozotocin-induced diabetic rats (Fig. 1). Subsequent experiments, therefore, determined if this decrease in aerobic glycolysis altered ouabain-sensitive 86Rb uptake in the diabetic blood vessels.


Figure 1
View larger version (24K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Glycolysis (µmol/g dry wt/h) and glucose oxidation (µmol/g dry wt/h) in aortic smooth muscle from control (n=16–19) and diabetic (n=21) rat. Values represent mean±s.e.m. for the rates of glycolysis and glucose oxidation during the second hour of the incubation. *P<0.05.

 
3.2 Ouabain-sensitive 86Rb uptake in vessels equilibrated in physiological salt solution
In the absence of endothelium, both ouabain-sensitive (active) and ouabain-insensitive (passive) 86Rb uptake was similar in aorta from control and insulin-deficient diabetic rats (Fig. 2). The presence of an intact endothelium decreased (P<0.05) ouabain-sensitive 86Rb uptake in aorta from streptozotocin-diabetic rats but had no effect on the ouabain-sensitive 86Rb uptake in aorta from non-diabetic control rats. There was no difference between the control and streptozotocin-diabetic rats in the presence of endothelium.


Figure 2
View larger version (23K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Ouabain-sensitive 86Rb uptake (µmol/g dry weight/min) in aortic smooth muscle from control and streptozotocin-diabetic rats with (n=5 per group) or without (n=16–21) intact endothelium. Values represent mean±s.e.m. *P<0.05.

 
In superior mesenteric artery (Fig. 3), ouabain-sensitive 86Rb uptake was similar in both groups. However, the ouabain-insensitive 86Rb uptake was slightly elevated (P<0.05) in the blood vessels from diabetic rats.


Figure 3
View larger version (13K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Ouabain-sensitive and ouabain-insensitive 86Rb uptake (µmol/g dry weight/min) in denervated superior mesenteric artery (SMA, n=7–9) from control and diabetic rat. Values represent mean±s.e.m. *P<0.05.

 
The metabolic consequences of streptozotocin-induced diabetes are associated with marked weight reduction and hypoinsulinemia, factors which may independently modify Na+/K+-ATPase activity [23, 24]. However, ouabain-sensitive 86Rb uptake (ouabain-sensitive 86Rb uptake = 0.425±0.05) was not altered by a comparable weight reduction (12-week weight: 345±5 g, n = 7) in food-restricted control rats or after administration of insulin for 10 weeks to streptozotocin-injected rats (ouabain-sensitive 86Rb uptake = 0.47±0.05, n = 7).

The next series of experiments evaluated the ouabain-sensitive 86Rb uptake when [Na]i and/or [K]o was altered in the presence or absence of specific metabolic substrates.

3.3 Ouabain-sensitive 86Rb uptake in Na-loaded aorta vessels at varying [K]o in the presence or absence of glucose or oxygen
Aortae were incubated in potassium-free PSS for 4 h which has been shown to inhibit the ouabain-sensitive 86Rb uptake over 90%25 and elevate Nai to approximately 150 mM. 86Rb uptake in the sodium-loaded vessels was subsequently measured at varying [K]O which resulted in minimal ([K]o = 1 mM) to maximal ([K]o = 10 mM) stimulation of ouabain-sensitive 86Rb uptake [13, 19, 25, 26]. Under these conditions, there was a linear increase in both the ouabain-sensitive and -insensitive 86Rb uptake in aorta (Fig. 4) and superior mesenteric artery (Table 2) as [K]o was increased. However, the increase in 86Rb uptake was the same for both control and streptozotocin-diabetic groups.


Figure 4
View larger version (18K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Ouabain-sensitive and ouabain-insensitive 86Rb uptake (µmol/g dry weight/min) in aortic smooth muscle from control (open symbols) and diabetic (closed symbols) rats equilibrated in K+-free PSS (circles), K+-free, glucose-free PSS (triangles) or K+-free, oxygen-free PSS (squares) followed by 86Rb uptake at varying [K]o. n for each group ranged from 5 to 11. Values represent mean±s.e.m.

 

View this table:
[in this window]
[in a new window]

  		Table 2 Ouabain-sensitive (active) and ouabain-insensitive (passive) 86Rb uptake (µm/g dry weight/min) in superior mesenteric artery from control and diabetic rats

 
Subsequent experiments evaluated ouabain-sensitive 86Rb uptake in Na-loaded vessels at [K]o 1 or 10 mM in the absence of glucose (Fig. 4; Table 2). Although the removal of glucose from the Na-loaded vessels decreased (from 18 to 24%) ouabain-sensitive 86Rb uptake during maximal stimulation ([K]o = 10 mM) in some groups (control aorta, P<0.01; diabetic SMA, P<0.05), at each [K]o, the 86Rb uptake in aorta or SMA was similar in control and insulin-deficient rats. The absence of glucose did not alter ouabain-insensitive 86Rb uptake at any [K]o.

The absence of oxygen from the Na-loaded vessels (but presence of glucose) resulted in an even greater reduction (from 29 to 47%) in the ouabain-sensitive 86Rb uptake during maximal stimulation in all groups (P<0.01). However, at each [K]o, the ouabain-sensitive 86Rb uptake in aorta (Fig. 4) or superior mesenteric artery (Table 2) was comparable in control and insulin-deficient rats. Oxygen deprivation did elevate the ouabain-insensitive 86Rb uptake in the SMA from diabetic animals at [K]o = 10 mM but had no effect on the ouabain-insensitive 86Rb uptake in the aorta.

3.4 Stimulation of ouabain-sensitive 86Rb uptake by insulin
A high concentration of insulin was required in these experiments: intima-media preparations are relatively insensitive to insulin stimulation [27], requiring a much higher insulin concentration than striated muscle to produce a comparable stimulation of glucose uptake [12]. Insulin increased ouabain-sensitive 86Rb uptake from 0.53±0.04 in PSS to 0.81±0.08 (P<0.05) in aorta from control animals (Fig. 5) but had no significant effect in aorta from insulin-deficient animals (0.60±0.04 in PSS and 0.69±0.05 in the presence of insulin). There was no effect of insulin on the ouabain-insensitive 86Rb uptake in either group.


Figure 5
View larger version (34K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Ouabain-sensitive 86Rb uptake (µmol/g dry weight/min) in aortic smooth muscle from control (C; n=10) and streptozotocin-diabetic (D; n=10) rats equilibrated in PSS. Values for ouabain-sensitive 86Rb uptake in PSS are the same as Fig. 2. Insulin (100 mU/ml) was added to the PSS during the last hour of the equilibration period. Values represent mean±s.e.m. *P<0.05.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
Decreased Na+/K+-ATPase activity has been reported in a variety of tissues from diabetic animals [28]although this has not always been a consistent finding [29]. There have also been reports that Na+/K+-ATPase activity was decreased in vascular smooth muscle from diabetic animals [14, 15]. However, in one of these studies, Na+/K+-ATPase activity was not decreased when evaluated in diabetic aorta without an intact endothelium [14], suggesting that endothelial rather than vascular factors mediate the inhibition of Na+/K+-ATPase activity in diabetic vascular smooth muscle. The results from the present study are consistent with this conclusion. The presence of an intact endothelium decreased the ouabain-sensitive 86Rb uptake in diabetic but not control aorta. However, in the absence of endothelium, ouabain-sensitive 86Rb uptake was similar in non-diabetic and diabetic aorta and SMA under unstimulated, physiological conditions (Figs. 2 and 3Go). The values for ouabain-sensitive rubidium uptake in diabetic rat aorta were comparable to the ouabain-sensitive rubidium [18]or potassium [30]uptake reported previously for non-diabetic rat aorta. Endothelium-mediated suppression of ouabain-sensitive 86Rb uptake in streptozotocin-diabetic rat aorta was similar to the results obtained in aorta from severely diabetic rabbits [14]. This latter study as well as others [31]has proposed a variety of mediators for this endothelial effect, including the arginine/nitric oxide pathway. Although severe weight reduction, fasting and the hypoinsulinemia associated with diabetes may potentially influence Na+/K+-ATPase activity [23, 24], neither a comparable weight reduction in control animals nor insulin administration, at a dose which completely restored left ventricular contractile function [32]in the diabetic rat heart, altered ouabain-sensitive 86Rb uptake.

The high rate of aerobic glycolysis that has been measured in the non-diabetic blood vessels studied thus far [9, 10, 12, 13], including rat aorta [8, 11], can supply most, if not all, of the ATP required to support basal Na+/K+-ATPase activity [11, 13]. The pathway(s) generating the ATP was not determined in the diabetic blood vessels. However, the comparable Na+/K+-ATPase activity in blood vessels from both groups indicates that, despite the significant decrease in glycolysis and glucose oxidation in diabetic aorta (Fig. 1), overall ATP production in the diabetic blood vessels was sufficient to support Na+/K+-ATPase activity under unstimulated, physiological conditions. However, the ability of aerobic glycolysis to completely support Na+/K+-ATPase activity in vascular smooth muscle is determined by the degree of stimulation. For example, Campbell and Paul [13]correlated aerobic glycolysis (using lactate production) and active potassium uptake in sodium-loaded, potassium-depleted porcine carotid arteries at varying [K]o and demonstrated that at [K]o 2–4 mM, ATP supplied by aerobic glycolysis was maximal. The increased Na+/K+-ATPase activity associated with further increases in [K]o >4 mM was supported by ATP generated via oxidative phosphorylation. Therefore, although basal Na+/K+-ATPase activity was not altered in insulin-deficient diabetic blood vessels, it was possible that the degree to which Na+/K+-ATPase could be stimulated was limited in the presence of impaired cellular metabolism in the diabetic tissue. Subsequent experiments were performed in the blood vessels from diabetic rats to determine if ATP production via aerobic glycolysis and/or oxidative metabolism could support Na+/K+-ATPase under conditions of maximal as well as minimal stimulation.

Ouabain-sensitive 86Rb uptake was similar in sodium-loaded aorta and superior mesenteric artery from diabetic and non-diabetic animals in the presence of both glucose and oxygen (Fig. 4; Table 2) under conditions of minimal ([K]o = 1 mM) to maximal ([K]o = 10 mM) stimulation. The 10- to 12-fold increase in 86Rb uptake (from basal unstimulated conditions to maximal stimulation of sodium-loaded vessels at [K]o = 10 mM) was similar to that achieved in rabbit carotid artery under similar conditions [19]and within the range of maximum transport estimated by 3H-ouabain binding [33–35]. These results suggest that when the substrates for both glycolytic and oxidative ATP production are available, maximal stimulation of Na+/K+-ATPase activity can be maintained in diabetic blood vessels. The one difference worth noting is that at [K]o = 1 mM, sodium-loading increased ouabain-sensitive rubidium uptake in rat aorta by 120–150% but had no effect on ouabain-sensitive rubidium uptake in superior mesenteric artery. It has been reported that the response of the superior mesenteric artery and aorta to inhibition of Na+/K+-ATPase was different [36], which may be related to the relative amount of Mg-ATPase in the two blood vessels [26].

Exogenous glucose appears to be the sole substrate for aerobic glycolysis [8, 10]. Therefore, in the absence of glucose, ATP in vascular smooth muscle would be supplied via oxidative metabolism. When experiments were repeated in glucose-free PSS, ouabain-sensitive rubidium uptake was similar in blood vessels from non-diabetic and diabetic rats (Fig. 4; Table 2). In both groups, glucose removal slightly decreased Na+/K+-ATPase activity during maximal stimulation ([K]O = 10 mM). These results suggest that, in the absence of glucose, oxidation of endogenous substrates, which appear to be primarily lipids [37]in vascular smooth muscle, can adequately support all ([K]O = 1 mM) or most ([K]O = 10 mM) of ouabain-sensitive uptake in both diabetic and non-diabetic aorta and superior mesenteric artery.

Repetition of these experiments in the absence of oxygen but in the presence of glucose further decreased Na+/K+-ATPase activity at [K]o = 1 and 10 mM (Fig. 4; Table 2). Thus, ATP production in rat aorta via anaerobic glycolysis cannot completely support Na+/K+-ATPase activity under conditions of either minimal ([K]o = 1 mM) or maximal ([K]o = 10 mM) stimulation. However, the decrease in Na+/K+-ATPase activity in the absence of oxygen was similar in both diabetic and non-diabetic blood vessels at all [K]o, which suggests that the impaired metabolism in the diabetic blood vessels did not differentially limit Na+/K+-ATPase activity under these conditions.

The results presented in the present study have indicated that Na+/K+-ATPase activity was similar in control and diabetic blood vessels when the ionic or substrate environment was manipulated. Insulin receptors have been identified in smooth muscle cells of rat aorta [27]and insulin has been shown to stimulate Na+/K+-ATPase in several tissues [38]including vascular smooth muscle [12]. The last series of experiments were undertaken to determine the effect of chronic diabetes on the insulin-receptor-mediated stimulation of Na+/K+-ATPase activity in diabetic rat aorta. As can be seen in Fig. 5, insulin stimulation of ouabain-sensitive rubidium uptake was significantly less in diabetic aorta. These results are consistent with previous reports that the response to insulin was diminished in diabetic tissues [27, 39].

In conclusion, these results indicate that, in the absence of endothelium, basal Na+/K+-ATPase activity in vascular smooth muscle from streptozotocin-diabetic rats is similar to that in non-diabetic blood vessels. Furthermore, the decrease in glycolysis and glucose oxidation in diabetic blood vessels does not limit the maximal stimulation of Na+/K+-ATPase activity. However, similar to reports in the literature [14], the presence of endothelium was associated with a marked decrease in Na+/K+-ATPase activity in aorta from the streptozotocin-diabetic rat. These results suggest that factors released from the endothelium of diabetic aorta subsequently suppress Na+/K+-ATPase activity of the underlying vascular smooth muscle.

Time for primary review 21 days.


   Acknowledgements
 
The authors would like to thank Ms. Ginger Batterton and Ms. Amy Dreisbach for their technical assistance with this manuscript.

This work was supported by NIH grant no. 1 R15 DK43913-01A1 and intramural funds provided by Midwestern University.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

  1. Arnqvist HJ. Influence of diabetes on metabolism of vascular smooth muscle. Acta Paediatr Scand Suppl 1977;270:106–111.
  2. Wertheimer HE, Ben-Tor V. Influence of diabetes on carbohydrate metabolism of aortic tissue. Diabetes 1962;11:422–425.
  3. Carlsson M, Arnquist HJ. Influence of exogenous substrates on adenosine triphosphate (ATP) concentration in normal and diabetic rat aorta in vitro. Acta Physiol Scand 1981;111:31–34.
  4. Paulson DJ, Crass MF. Endogenous triacylglycerol metabolism in diabetic heart. Am J Physiol 1982;242:H1084–H1094.
  5. Randle PJ, Kerbey AL, Espinnal J. Mechanisms decreasing glucose oxidation in diabetes and starvation: role of lipid fuels and hormones. Diab Metab Rev 1988;4:623–638.
  6. Ruderman NB, Williamson JR, Brownlee M. Glucose and diabetic vascular disease. FASEB J 1992;6:2905–2914.
  7. Wall SR, Lopaschuk GD. Glucose oxidation rates in fatty acid-perfused isolated working hearts from diabetic rats. Biochim Biophys Acta 1989;1006:97–103.
  8. Kutchai H, Geddis LM. Regulation of glycolysis in rat aorta. Am J Physiol 1984;247:C107–C114.
  9. Paul RJ. Chemical energetics of vascular smooth muscle. In: Bohr DH, Somlyo AP, Sparks HV, eds. Handbook of Physiology, Section 2, The Cardiovascular System, Vascular Smooth Muscle VII. Washington: American Physiological Society, 1980;201–235.
  10. Paul RJ. Smooth muscle energetics. Annu Rev Physiol 1989;51: 331–349.
  11. Paul RJ. Functional compartmentalization of oxidative and glycolytic metabolism in vascular smooth muscle. Am J Physiol 1983;244:C399–C409.
  12. Lynch RM, Paul RJ. Compartmentation of carbohydrate metabolism in vascular smooth muscle. Am J Physiol 1987;252:C328–C334.
  13. Campbell JD, Paul RJ. The nature of fuel provision for the Na+-K+-ATPase in porcine vascular smooth muscle. J Physiol 1992;447:67–82.
  14. Gupta S, Sussman I, McArthur CS, Tornheim K, Cohen RA, Ruderman NB. Endothelium-dependent inhibition of Na+K+ ATPase activity in rabbit aorta by hyperglycemia. J Clin Invest 1992;90:727–732.
  15. Ohara T, Sussman KE, Draznin B. Effect of diabetes on cytosolic free Ca2+ and Na+-K+-ATPase in rat aorta. Diabetes 1991;40:1560–1563.
  16. Burnstock G. Structural characteristics, mechanisms of contraction, innervation and proliferation of smooth muscle cells. In: Wolf S, Werthess NT, eds. The Smooth Muscle of the Artery. New York: Plenum Press, 1975;20–29.
  17. Aprigliano O, Hermsmeyer K. In vitro denervation of the portal vein and caudal artery of the rat. J Pharmacol Exp Ther 1976;198:568–571.
  18. Smith JM, Sanchez AA, Jones AW. Comparison of rubidium-86 and potassium-42 fluxes in rat aorta. Blood Vessels 1986;23:297–309.
  19. Heidlage JR, Jones A.W. The kinetics of ouabain-sensitive ionic transport in the rabbit carotid artery. J Physiol 1981;317:243–262.
  20. Kobayashi K, Neely JR. Control of maximum rates of glycolysis in rat cardiac muscle. Circ Res 1979;44:166–175.
  21. Arnqvist HJ. Effects of increasing glucose concentrations on the glucose metabolism in arterial tissue and intestinal smooth muscle. Acta Physiol Scand 1973;88:481–490.
  22. Lynch R, Paul R. Glucose uptake in porcine carotid artery: relation to alterations in active Na+-K+ transport. Am J Physiol 1984;247:C433–C440.
  23. Kjeldsen K, Norgaard A, Clausen T. Effects of ouabain, age and K-depletion on K-uptake in rat soleus muscle. Pflüger's Arch 1985;404:365–373.
  24. Nishida K, Ohara T, Johnson J, et al. Na+/K+ ATPase activity and its {alpha}II subunit gene expression in rat skeletal muscle: influence of diabetes, fasting, and refeeding. Metabolism 1992;41:56–63.
  25. Jones AW. Kinetics of active sodium transport in aorta from control and deoxycorticosterone hypertensive rats. Hypertension 1981;3:631–640.
  26. Jones AW. Content and fluxes of electrolytes. In: Bohr DH, Somlyo AP, Sparks HV, eds. Handbook of Physiology, Section 2, The Cardiovascular System, Vascular Smooth Muscle VII. Washington: American Physiological Society, 1980;201–235.
  27. Capron L, Philippe M, Fiessinger JN, Housset E. Diabetes and insulin: actions and interactions upon glucose metabolism of the rat aorta. Diab Metab 1984;10:78–84.
  28. Kjeldsen K, Braendgaard H, Sidenius P, Larsen JS, Horgaard A. Diabetes decreases Na+-K+ pump concentration in skeletal muscles, heart ventricular muscle, and peripheral nerves of rat. Diabetes 1987;36:842–848.
  29. Reddi AS, Dasmahapatra A, Jyothirmayi GN, Jayasundaramma B. Erythrocyte Ca, Na/K-ATPase in long-term streptozotocin diabetic rats. Am J Hypertens 1992;5:863–868.
  30. Magliola L, McMahon EG, Jones A. Alterations in active Na-K transport during mineralocorticoid-salt hypertension in the rat. Am J Physiol 1986;250:C540–C546.
  31. Poston L, Taylor PD. Endothelium-mediated vascular function in insulin-dependent diabetes mellitus. Clin Sci 1995;88:245–255.
  32. Romano FD, Kopp SJ, Daar JT, Smith CA. The antiadrenergic effect of cyclopentyladenosine on myocardial contractility is reduced in vivo in diabetic rats. Can J Physiol Pharmacol 1994;72:1245–1251.
  33. Aalkjaer C, Kjeldsen K, Norgaard A, Clausen T, Mulvany MJ. Ouabain binding and Na+ content in resistance vessels and skeletal muscles of spontaneously hypertensive rats and K+-depleted rats. Hypertension 1985;7:277–286.
  34. Clausen T, Everts ME, Kjeldsen K. Quantification of the maximum capacity for active sodium-potassium transport in rat skeletal muscle. J Physiol 1987;388:163–181.
  35. Deth RC, Smart JL, Lynch CJ, Walsh R. Lack of correlation between [3H]ouabain binding and Na-K ATPase inhibition in rat aorta. Eur J Pharmacol 1984;99:45–55.
  36. Mulvany MJ, Aalkjaer C, Hilsson H, Korsgaard N, Petersen T. Raised intracellular sodium consequent to sodium-potassium-dependent ATPase inhibition does not cause myogenic contractions of 150 µM arteries from rat and guinea pig. Clin Sci 1982;63:45s–48s.
  37. Odessey R, Chace K. Utilization of endogenous lipid, glycogen and protein by rabbit aorta. Am J Physiol 1982;243:H128–H132.
  38. Rosic NK, Standaet ML, Pollet RJ. The mechanism of insulin stimulation of (Na+,K+)-ATPase transport activity in muscle. J Biol Chem 1985;260:6206–6212.
  39. Sowers JR, Levy J, Zemel MB. Hypertension and diabetes. Med Clin North Am 1988;72:1399–1414.

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J. Appl. Physiol.Home page
C. A. Witczak and M. Sturek
Exercise prevents diabetes-induced impairment in superficial buffer barrier in porcine coronary smooth muscle
J Appl Physiol, March 1, 2004; 96(3): 1069 - 1079.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Smith, J. M
Right arrow Articles by M. Solar, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Smith, J. M
Right arrow Articles by M. Solar, S.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?