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(Circulation Research. 1997;81:996-1004.)
© 1997 American Heart Association, Inc.

Articles

Increased Myogenic Tone and Diminished Responsiveness to ATP-Sensitive K⁺ Channel Openers in Cerebral Arteries From Diabetic Rats

Paul A. Zimmermann¹, Harm J. Knot¹, Andrá S. Stevenson, , Mark T. Nelson

From the Department of Pharmacology (A.S.S., H.J.K., M.T.N.) and the Division of Cardiology (P.A.Z.), The University of Vermont, Burlington.

Correspondence to Dr Mark T. Nelson, Department of Pharmacology, The University of Vermont, Given Building, Room B303, Burlington, VT 05405-0068. E-mail nelson{at}northpole.med.uvm.edu

	Abstract

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Abstract Diabetes mellitus has profound adverse effects on vascular and, in particular, endothelial function. Although pressure-induced constriction ("myogenic tone") is a major contributor to the regulation of blood flow, little is known about the effects of diabetes on this response. Diabetes has been shown to diminish the dilation of cerebral arteries to synthetic ATP-sensitive K⁺ (K_ATP) channel openers. In this study, we explored the effects of diabetes induced in rats by streptozotocin on cerebral artery (250 to 300 µm) myogenic tone and on vasodilations to the synthetic K_ATP channel openers pinacidil and levcromakalim. Elevation of intravascular pressure caused a graded membrane potential depolarization and constriction, which was greater in arteries from diabetic rats compared with normal rats (at 60 mm Hg, 5 mV more depolarized and 22 µm more constricted). Pressurized arteries (at 60 mm Hg) from diabetic rats were 5- to 15-fold less sensitive to pinacidil and levcromakalim than were control arteries (EC₅₀ values for pinacidil and levcromakalim were 1.4 and 0.6 µmol/L, respectively, in diabetic animals and 0.3 and 0.04, respectively, in control animals; P<.05). Removal of the endothelium or addition of a NO synthase inhibitor, N^G-nitro-L-arginine (LNNA), in control arteries decreased the sensitivity to K_ATP channel openers and depolarized and constricted control arteries to levels similar to those observed in arteries from diabetic animals. Sodium nitroprusside caused a membrane potential hyperpolarization and enhanced the response to pinacidil in arteries from diabetic animals. Removal of the endothelium or LNNA had little effect on the apparent K_ATP channel opener sensitivity, the membrane potential, and pressure-induced constrictions of arteries from diabetic animals. The results are consistent with the hypothesis that this type of diabetes leads to a decrease in tonic NO release from the endothelium, which in turn causes membrane potential depolarization and vasoconstriction, resulting in a diminished response to K_ATP channel openers.

Key Words: diabetes • K⁺ channel • endothelium • vascular smooth muscle • nitric oxide

	Introduction

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Diabetes mellitus is a devastating disease that affects 5% of the American population. There is a 2- to 4-fold increase in the risk of coronary heart disease, cerebrovascular disease, congestive heart failure, and other cardiovascular complications due to diabetes.^1,2 The underlying mechanisms causing this increased risk remain unclear. Abnormalities in lipid metabolism, coagulation cascade, and glycation products may contribute to increased vascular disease in diabetes.³

Recent evidence suggests that diabetes causes abnormal endothelial function.^4–7 For example, endothelium-dependent dilations of cerebral arteries and arterioles are profoundly affected by diabetes.^8–10 Endothelium dysfunction during diabetes appears to be a major link in the pathogenesis of vascular disease.^11,12

Changes in vascular smooth muscle function associated with diabetes are well documented.^13,14 Of particular interest, Mayhan and Faraci¹⁵ (1993) and Mayhan¹⁶ (1994) recently demonstrated that cerebral arteries from diabetic rats dilate less than control arteries to the K_ATP channel openers aprikalim and levcromakalim. These studies suggested that part of the functional change occurring in vascular smooth muscle during diabetes may be directly related to alterations in ion channel function.

Elevation of intravascular pressure causes a graded membrane potential depolarization and contraction (myogenic tone) of the smooth muscle cells in intact cerebral arteries.^17–19 Myogenic tone is a major contributor to the regulation of arterial diameter.²⁰ Myogenic tone of small cerebral arteries depends on Ca²⁺ entry through voltage-dependent Ca²⁺ channels,²¹ since Ca²⁺ channel blockers or membrane potential hyperpolarization through activation of K⁺ channels causes vasodilation.^12,19,22,23 Hypoxia,^24,25 calcitonin gene-related peptide,^26–29, adenosine,²⁹ and synthetic K⁺ channel openers (eg, pinacidil, levcromakalim, and aprikalim)^29,30 appear to dilate cerebral arteries, in part, through membrane potential hyperpolarization caused by the activation of K_ATP channels.

The first goal of the present study was to determine the effect of diabetes on pressure-induced changes in diameter and membrane potential in rat cerebral arteries. The second goal of this study was to explore the mechanisms for the diminished sensitivity of cerebral arteries to synthetic K_ATP channel openers. Our results are consistent with the hypothesis that diabetes mellitus reduces the tonic release of NO from the endothelium, leading to membrane potential depolarization and vasoconstriction. This decrease of NO release and/or the subsequent membrane potential depolarization causes a decrease in the sensitivity of the arteries to K_ATP channel openers. These findings have implications for the regulation of arterial diameter by pressure, NO, and K_ATP channel openers during diabetes.

	Materials and Methods

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Induction of Diabetes and Preparation
Female Sprague-Dawley rats (12 weeks old, 280 g) were studied. Diabetes was induced with a single intraperitoneal injection of STZ (60 mg/kg) dissolved in 1 mL sterile saline as a vehicle. Control rats received a single intraperitoneal injection of 1 mL sterile saline solution. Induction of diabetes was confirmed by occurrence of glucosuria on 2 consecutive days after the intraperitoneal injection. Blood samples were obtained when the animals were killed for study, between 4 to 8 weeks after the development of glucosuria. Blood glucose measurements were determined by using a glucoscan meter (One Touch II, Lifescan Inc).

Female Sprague-Dawley rats were euthanized with pentobarbital (130 mg/kg IP) and killed by thoractomy. The chest was opened, and a blood glucose sample was obtained. The animals were decapitated, and the brains were removed and quickly transferred into normal PSS (for composition, see below) at 4°C. Resistance-sized proximal middle cerebral arteries were isolated and dissected from the surrounding connective tissue. These cerebral arteries were then cannulated and mounted in an arteriograph. Intravascular pressure was gradually increased in 20 mm Hg steps, with the vessel being perfused in PSS (for composition, see below) at 37°C, after a 15-minute equilibration period. Concentration-response curves to various vasodilators were performed at an intraluminal pressure of 60 mm Hg, the estimated pressure experienced by the vessel in vivo (estimated to be 50% of systolic blood pressure).

Maximal passive diameter of pressurized arteries was determined as the arterial diameter in Ca²⁺-free PSS. The arteries were denuded of endothelium by placing an air bubble in the lumen for 1 minute, followed by a luminal wash with distilled water for 30 seconds. Endothelial disruption was verified by the absence of a dilator response to acetylcholine after myogenic tone had developed.

Recording Methods
Arterial diameter was measured with a video dimension analyzer (Living Systems Instrumentations). Membrane potential was recorded in intact pressurized arteries, using conventional intracellular glass microelectrodes filled with 3 mol/L KCl solution and tip resistances of 40 to 60 M. Smooth muscle cells were impaled from the cleaned adventitial side of the pressurized artery. Membrane potentials were measured with an AXOCLAMP 2B amplifier (at 0.5- to 1-kH bandwidth, Axon Instruments Inc), and data were recorded using an Axotape 2.0/Digidata 1200 data aquisition system (Axon Instruments Inc) on a Gateway 386/20DX PC. Criteria for acceptance of recordings were as follows: (1) an abrupt change in potential on impalement of cells, (2) stable membrane potential for at least 2 minutes before experimental manipulations, (3) maintained impalement throughout the experimental protocol, and (4) unchanged tip resistance before and after impalement and tip potentials of <3 mV.

Solutions and Drugs
A PSS was used as the bathing solution and had the following composition (mmol/L): NaCl 119, KCl 4.7, NaHCO₃ 24.0, KH₂PO₄ 1.2, CaCl₂ 1.6, MgSO₄ 1.2, EDTA 0.023, and glucose 11.0 (pH 7.4). This solution was continuously bubbled with 95% O₂/5% CO₂. The arteries were superfused at 3 to 6 mL/min (bath volume, 6 mL). All intact vessel experiments were performed at 37°C under continuous superfusion with PSS. All chemicals and reagents were from Sigma Chemical Co unless otherwise specified. Pinacidil and levcromakalim were obtained from Research Biochemicals International.

Data Analysis and Presentation
The approximation of the half effective concentration (EC₅₀) of drug giving a half-maximal response (eg, vasodilation) was calculated from fitting a logistical equation (sigmoidal nonlinear least-squares fit) to the concentration response curves using the ORIGIN program (Microcal Software Inc). Vessel distension ratio (D_R) values were obtained from passive (0 mmol/L Ca²⁺ PSS) arterial diameter measurements and were calculated using the following equation: D_R=D_x/D₀, where D_x is the diameter at a given pressure x, and D₀ represents arterial diameter at 0 pressure obtained through extrapolation of a third-order polynomial fitted to the passive pressure/diameter curves for each artery.³¹ Membrane potential values are expressed in millivolts as mean±sample SD from n different arteries. Diameter values are expressed in microns as mean±SEM for n vessels. Statistical significance was tested at the 95% (P<.05) confidence level using Student's paired t test on independent measurements. Asterisks on figures indicate significant difference from control values.

	Results

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Rats were euthanized at 16 to 20 weeks of age 4 to 8 weeks after STZ treatment. The average body weight of the rats at 12 weeks was 280 g. Body weight was randomly measured in the two groups at the time of euthanasia. The diabetic rats had an average body weight of 280±25 g (n=17). The average body weight of control animals (saline-injected) was 337±24 g (n=11). The average blood glucose concentration was significantly higher in diabetic rats compared with control rats: 17.6±0.6 mmol/L (317±10 mg/dL) (n=31) and 4.6±0.2 mmol/L (83±4 mg/dL) (n=47), respectively (P<.05). The mean proximal middle cerebral arterial diameter at 10 mm Hg was 169±4 µm in control rats (n=33) and 181±3 µm in diabetic rats (n=22) (P<.05) (Fig 1A and 1B). The mean maximal diameters at 100 mm Hg for control rats and diabetic rats were 257±5 µm (n=33) and 275±5 µm (n=22), respectively (P<.05) (Fig 1A and 1B).

View larger version (22K): [in this window] [in a new window]   Figure 1. Arteries from diabetic animals and denuded control arteries constrict more to pressure than do control arteries from normal animals with intact endothelium. Original recordings of the arterial diameter response to increasing intravascular pressure in middle cerebral arteries from rats are shown. Increasing intravascular pressure constricts cerebral arteries (lower traces in panels A to C). Passive responses of arterial diameter to pressure (measured in nominal Ca2+-free buffer) are shown in the top traces of panels A to C. A, Vessel from a normal rat (C) with intact endothelium. B, Artery from a diabetic animal (DM) with intact endothelium. C, Vessel obtained from a normal rat from which the endothelium had been removed (C-ENDO). The vertical dashed lines indicate pressure steps in the individual panels following the scale below.

Pressure-Induced Constrictions Are Greater in Cerebral Arteries From Diabetic Than Control Rats
Cerebral arteries from both control and diabetic rats constricted in response to a graded increase in pressure (Figs 1 and 2). As pressure was increased into the physiological range that these arteries experience in vivo, estimated at 60 mm Hg or higher (50% of systolic pressure), arteries from diabetic rats constricted more than control arteries (compare Figs 1A and 1B, and see Fig 2). At 60 mm Hg, arteries from diabetic rats constricted by 85±5 µm (32±2%) (n=8) compared with 63±2 µm (27±1%) (n=6) in control arteries (P<.05) (Fig 2). At 100 mm Hg, arteries from diabetic rats constricted by 112±6 µm (39±2%) (n=8) compared with 73±2 µm (30±1%) (n=6) in control arteries (P<.05) (Figs 1 and 2).

View larger version (20K): [in this window] [in a new window]   Figure 2. Arterial constriction as a function of transmural pressure in control (C) and diabetic (DM) arteries, with and without endothelium (-ENDO). Pressure-induced constriction ("myogenic tone") in arteries is calculated as the difference in arterial diameter in PSS with and without Ca2+. Arteries from diabetic rats constricted significantly more to pressures of 60 mm Hg compared with arteries from control animals. Removal of the endothelium from arteries obtained from control animals [C(-ENDO)] increased the pressure-induced constriction, effectively eliminating the difference in tone between control and diabetic arteries, whereas removal of the endothelium in arteries from diabetic rats [DM(-ENDO)] had no effect. *P<.05 vs control conditions.

Since endothelial function is altered in cerebral arteries from diabetic animals,^8–10 the effects of the removal of the endothelium on pressure-induced constrictions were examined. Arteries from control animals constricted more to pressure after the removal of the endothelium (Figs 1C and 2). Denuded arteries from control rats constricted by 84±10 µm (35±4%) (n=6) at 60 mm Hg and by 110±10 µm (42±5%) (n=6) at 100 mm Hg (Fig 1C) compared with 63 and 73 µm in arteries with intact endothelium (P<.05). Denuded arteries from diabetic rats had no significant increase in tone compared with nondenuded arteries (Fig 2). Denuded arteries from diabetic rats constricted by 86±5 µm (33±2%) (n=7) at 60 mm Hg and 110±6 µm (39±2%) (n=7) at 100 mm Hg (Fig 2). These results suggest that endothelial cells from control arteries tonically release a dilating factor and that diabetes disrupts this process.

To exclude the possibility that the diabetic state had influenced the passive properties of the arteries, the passive properties of the arteries from control and diabetic animals, with and without intact endothelium, were examined in nominal Ca²⁺-free buffer. Calculated distension ratios were similar at different levels of intravascular pressure under all conditions (Fig 3). Therefore, the observed difference in pressure-induced constriction between diabetic and control animals does not appear to be a consequence of structural alterations in vessel stiffness.

View larger version (16K): [in this window] [in a new window]   Figure 3. Diabetes and removal of the endothelium do not alter a passive property (distension ratio) of myogenic cerebral arteries. Passive properties of cerebral arteries from control and diabetic animals with intact endothelium (C and DM groups, respectively) and with denuded endothelium [C(-ENDO) and DM(-ENDO) groups, respectively]. There were no significant differences in the passive dilatory responses to pressure between arteries from diabetic vs control animals. Removal of the endothelium did not change the passive dilation of the arteries to pressure.

Acetylcholine and SNP Responses in Cerebral Arteries From Control and Diabetic Rats
To assess the function of the endothelium, we studied the vasodilator response to acetylcholine, an endothelium-dependent vasodilator, in cerebral arteries from control and diabetic rats pressurized to 60 mm Hg (Fig 4). Acetylcholine-induced dilations were markedly reduced in cerebral arteries from diabetic rats (Fig 4A). Acetylcholine (10 µmol/L) dilated arteries from diabetic animals by 7±13 µm (n=6) compared with 42±5 µm (n=6) in control tissues (P<.05). The diminished response of cerebral arteries to acetylcholine from diabetic rats is consistent with previous observations by others^9,32,33 and supports the hypothesis that endothelial cells are dysfunctional in diabetes.

View larger version (16K): [in this window] [in a new window]   Figure 4. Diabetes reduces endothelium-dependent dilations of cerebral arteries to acetylcholine (Ach) but not to the nitrovasodilator SNP. A, Response of arteries from control (C) and diabetic (DM) animals to the endothelium-dependent vasodilator Ach (10 µmol/L) at an intravascular pressure of 60 mm Hg is shown. B, Dilations to SNP (100 nmol/L and 100 µmol/L) were similar in control arteries, control arteries without endothelium (-Endo), and diabetic arteries (n=4 or 5). The dilations were not statistically different (P<.05).

Our results suggest that diabetes interrupts basal NO release from the endothelium.³⁴ To explore a possible direct effect of diabetes on smooth muscle cell responsiveness to NO, the dilator effects of the NO donor SNP were examined. SNP fully relaxed arteries from control and diabetic animals (Fig 4B). SNP at 100 nmol/L dilated control arteries (±endothelium) and arteries from diabetic animals to a similar extent (Fig 4B). These results suggest that diabetes did not significantly alter smooth muscle responsiveness to SNP.

Dilations to K_ATP Channel Openers Are Reduced in Diabetes
Vasodilations of cerebral arterioles to the K_ATP channel openers aprikalim and levcromakalim are reduced in diabetes.^15,16 We extended this observation by demonstrating that dilations to other types of K_ATP channel openers, such as pinacidil,²⁹ are also reduced in diabetes (Figs 5 and 6). Dilations to pinacidil and levcromakalim of pressurized (to 60 mm Hg) cerebral arteries (±endothelium) from control and diabetic animals were measured. Cerebral arteries from diabetic rats were less sensitive to these K_ATP channel openers. For example, pinacidil at 0.3 µmol/L dilated arteries from diabetic animals by only 5±4% compared with a dilation of 34±8% in the control animals (P<.05). Similarly, levcromakalim at 0.3 µmol/L dilated arteries from diabetic animals by 20±5% compared with an almost maximal dilation of 76±6% in arteries from control animals (P<.05). Both pinacidil and levcromakalim dilated arteries from control and diabetic animals to a maximum of 80±5% (n=18). The half-maximal effective concentration (EC₅₀) for dilation to pinacidil in arteries from diabetic rats was 1.4±0.1 µmol/L (n=4) compared with 0.3±0.1 µmol/L (n=4) in control rats (P<.05), indicating a 5-fold decrease in sensitivity (Fig 5A). Similarly, EC₅₀ for levcromakalim dilation in arteries from diabetic rats was 0.6±0.1 µmol/L (n=5) compared with 0.04±0.01 µmol/L (n=5) in control rats (P<.05), a 15-fold decrease in sensitivity (Fig 6A).

View larger version (20K): [in this window] [in a new window]   Figure 5. Diabetes or endothelium removal of control arteries reduces the dilatory responsiveness of cerebral arteries to vasodilation by the synthetic KATP channel opener pinacidil. A, Concentration-response curves for control animals with an intact endothelium (C) and diabetic animals with an intact endothelium (DM). B, Concentration-response curves for control arteries from which the endothelium had been removed [C(-ENDO)] and diabetic arteries from which the endothelium had been removed [DM(-ENDO)]. Removal of the endothelium did not affect pinacidil-induced relaxations of diabetic animals, but this maneuver abolished the difference between control and diabetic arteries. % vasodilation reflects the percent reversal of the pressure-induced constriction of the arteries at an intravascular pressure of 60 mm Hg. *P<.05 vs control conditions.

View larger version (20K): [in this window] [in a new window]   Figure 6. Diabetes or endothelium removal of control arteries reduces the dilatory responsiveness of cerebral arteries to vasodilation by the synthetic KATP channel opener levcromakalim. A, Concentration-response curves for control animals with an intact endothelium (C) and diabetic animals with an intact endothelium (DM). B, Concentration-response curves for control arteries from which the endothelium had been removed [C(-ENDO)] and diabetic arteries from which the endothelium had been removed [DM(-ENDO)]. Removal of the endothelium did not affect levcromakalim-induced relaxations of diabetic animals, but this maneuver abolished the difference between control and diabetic arteries. % vasodilation reflects the percent reversal of the pressure-induced constriction of the arteries at an intravascular pressure of 60 mm Hg. *P<.05 vs control conditions.

Dilations to K_ATP Channel Openers Are Reduced by Endothelium Removal or by Inhibitors of NO Synthase
Loss of endothelial function appears to be an important contributor to enhanced pressure-induced "myogenic" vasoconstriction during diabetes (Fig 1). We thus sought to examine the effects of endothelium removal on dilations to pinacidil and levcromakalim (Figs 5B and 6B). Removal of the endothelium from control arteries reduced the sensitivity of the pressurized arteries to pinacidil and levcromakalim to that observed in arteries from diabetic animals. For example, pinacidil at 0.3 µmol/L dilated arteries from control animals from which the endothelium had been removed by 17±6% compared with a dilation of 34±8% in the control arteries. Similarly, levcromakalim at 0.3 µmol/L dilated denuded arteries from control animals by 25±9% compared with an almost maximal dilation of 76±6% in control arteries. Both pinacidil and levcromakalim dilated denuded arteries from control and diabetic animals to a maximum of 88±5% (n=22). The EC₅₀ for pinacidil dilation of denuded arteries from control rats shifted rightward from 0.3±0.1 to 1.0±0.1 µmol/L (n=4), and similarly, the EC₅₀ for levcromakalim dilation shifted from 0.04±0.01 to 0.7±0.1 µmol/L (n=6), respectively (P<.05) (Figs 5B and 6B). In support of the change in K_ATP channel opener sensitivity with diabetes being mediated by the endothelium, denuded arteries from control and diabetic animals dilated to the same extent to pinacidil (Fig 5B) and levcromakalim (Fig 6B). None of these values were statistically different (in denuded arteries from diabetic rats: EC₅₀, 1.1±0.1 µmol/L, n=5, for pinacidil and 0.4±0.1 µmol/L, n=7, for levcromakalim).

The above data suggested that the effectiveness of K_ATP channel activation could be diminished by decreasing the release of a relaxing factor from the endothelium. Since NO released from the endothelium can cause vasodilation, the effects of pinacidil were assessed in the presence of LNNA (100 µmol/L), a NO synthase inhibitor (Fig 7). LNNA constricted pressurized (at 60 mm Hg) cerebral arteries from control animals by 35±7 µm (n=11) (P<.05), whereas LNNA did not have an effect on arteries from diabetic animals (13±13 µm, n=5). LNNA significantly reduced the sensitivity of pressurized control arteries to pinacidil, whereas it had little effect on arteries from diabetic animals (Fig 7). For example, pinacidil at 0.3 µmol/L dilated arteries from control animals in the presence of 100 µmol/L LNNA by 2±1% (n=11) compared with a dilation of 34±8% (P<.05) in the control arteries. Pinacidil at 0.3 µmol/L dilated arteries from diabetic animals in the presence of 100 µmol/L LNNA by 4±4% (n=5) compared with a dilation of 5±4% in the control diabetic arteries. Pinacidil dilated arteries from control and diabetic animals in the presence of 100 µmol/L LNNA to a maximum of 83±4% (n=16). The EC₅₀ for pinacidil dilation of arteries treated with 100 µmol/L LNNA from control rats shifted rightward from 0.3±0.06 to 1.5±0.05 µmol/L (n=8) (P<.05). The concentration-response curves to pinacidil for LNNA-treated control arteries, LNNA-treated diabetic arteries, and diabetic arteries were not statistically different (Fig 7). Further, LNNA did not significantly alter pinacidil-induced dilations of endothelium-denuded pressurized arteries from control or diabetic animals (EC₅₀, 1.3±0.4 µmol/L and 1.5±0.9 µmol/L, respectively). These results indicate that the difference in the apparent K_ATP channel opener sensitivity is due to reduced release of endothelium-derived NO affecting the arterial smooth muscle cells.

View larger version (20K): [in this window] [in a new window]   Figure 7. Diabetes-induced reduction in responsiveness of cerebral arteries to the KATP channel opener pinacidil is mimicked by the application of the NO synthase inhibitor LNNA to control arteries with intact endothelium (C), whereas LNNA has no effect on the pinacidil response in diabetic arteries with intact endothelium (DM). Shown are concentration-response curves to the synthetic KATP channel opener pinacidil in arteries from C and DM groups in the absence (curves replotted from Fig 5A) and presence [C(+LNNA) and DM(+LNNA), respectively] of the NO synthase inhibitor LNNA (100 µmol/L). % vasodilation reflects the percent reversal of the pressure-induced constriction of the arteries at an intravascular pressure of 60 mm Hg. *P<.05 vs control conditions.

In addition, in the absence of K_ATP channel openers, K_ATP channels do not appear to contribute significantly to the regulation of myogenic tone, since a K_ATP channel blocker, GLIB (10 µmol/L),^22,29 was without effect on arteries from normal and diabetic animals. Arterial diameters (at 60 mm Hg) were similar in PSS and in PSS+10 µmol/L GLIB for normal rats (change was +2±35 µm, n=5) and in PSS and in PSS+10 µmol/L GLIB for diabetic rats (change was -4±26 µm, n=7), respectively.

LNNA Depolarizes the Membrane Potential of Smooth Muscle Cells in Pressurized Arteries of Control, but Not Diabetic, Rats
Since membrane potential depolarization can constrict arteries and a number of endothelium-relaxing factors, including NO, cause membrane potential hyperpolarization, we explored the possibility that arteries from diabetic animals were depolarized compared with arteries from control animals. To investigate this issue, membrane potentials of pressurized (to 60 mm Hg) arteries from control and diabetic rats were measured in the presence and absence of LNNA, with and without GLIB present. Pressurized (60 mm Hg) arteries from control animals had membrane potentials of -45.6±1.2 mV (n=12) in PSS and -45.4±1.3 mV (n=5) in PSS+10 µmol/L GLIB. Elevating intravascular pressure from 10 to 60 mm Hg caused a membrane potential depolarization from -60 to -40 mV, as previously described.^17–19 Pressurized (60 mm Hg) arteries from diabetic animals were 5 mV more depolarized and had membrane potentials of -40.3±1.6 mV (n=15) (P<.05) in PSS and -40.3±1.5 mV (n=6) (P<.05) in PSS+10 µmol/L GLIB. The addition of LNNA (50 µmol/L) caused a 5-mV depolarization in arteries from control animals (to -40.5±1.9 mV, n=6, P<.05, Fig 8A), but it did not alter the membrane potential of arteries from diabetic animals (-39.5±1.5 mV, n=6, Fig 8B). Similarly, in the presence of 10 µmol/L GLIB, the addition of LNNA depolarized arteries from control animals by 4 to 5 mV to -41.2±1.2 mV (n=6), whereas it had no significant effect on membrane potentials in arteries from diabetic animals (-40.3±1.5 mV, n=6). These data indicate that GLIB (10 µmol/L) had no effect on membrane potentials of arteries from normal and diabetic rats, consistent with the observed lack of effect of GLIB on arterial diameter and, taken together, suggest that K_ATP channels do not contribute significantly to the arterial membrane potential under these conditions. Furthermore, GLIB did not alter the depolarizing response to LNNA in arteries from control animals. These results are consistent with the idea that tonic release of NO from the endothelium of control arteries causes a membrane potential hyperpolarization and dilation, independent of K_ATP channels.

View larger version (27K): [in this window] [in a new window]   Figure 8. Membrane potential measurements in pressurized (to 60 mm Hg) middle cerebral arteries from control (C) and diabetic (DM) rats. Cerebral arteries from the DM group are more depolarized compared with arteries from the C group. A and B, The NO synthase inhibitor LNNA (50 µmol/L) depolarized smooth muscle cells in arteries from the C group (panel A), whereas the addition of the KATP channel inhibitor GLIB (10 µmol/L) had no effect (panel B). Vm indicates membrane voltage. C and D, LNNA was without effect on the membrane potential of smooth muscle cells in arteries from the DM group (panel C). GLIB (10 µmol/L) was also without effect when given either alone (panel D) or after application of LNNA (panel C).

SNP Hyperpolarizes the Membrane Potential and Restores Pinacidil Sensitivity
Consistent with the tonic release of NO causing a membrane potential hyperpolarization in control arteries, SNP (100 nmol/L) caused a membrane potential hyperpolarization of diabetic arteries from -40.3±1.6 to -46.1±1.2 mV, or by 5.8 mV (n=7) (P<.001) (Fig 9A). GLIB did not affect the SNP-induced hyperpolarization of these arteries; in the presence of GLIB, SNP hyperpolarized the arteries to -47.3±1.0 mV (n=4). SNP (100 nmol/L) caused a membrane potential hyperpolarization of normal arteries from -45.7±1.2 to -48.2±0.8 mV (n=6), or by 2.5 mV (P<.001) (n=6). As predicted from the proposed model in Fig 10, pinacidil (300 nmol/L)–induced dilations of pressurized arteries from diabetic animals or control arteries denuded of their endothelium were enhanced by SNP. Pinacidil (300 nmol/L) dilated pressurized (60 mm Hg) arteries from diabetic animals by 5±4% (n=4) and by 28±6% (n=5) in the presence of SNP (100 nmol/L). Pinacidil (300 nmol/L) dilated control arteries, denuded control arteries, and denuded control+SNP arteries by 34±8% (n=4), 17±6% (n=4), and 30±2% (n=4), respectively (Fig 9B).

View larger version (16K): [in this window] [in a new window]   Figure 9. A, SNP (100 nmol/L) hyperpolarizes the membrane potential of smooth muscle cells in pressurized (60 mm Hg) cerebral arteries from DM rats. Vm indicates membrane voltage. B, SNP increases pinacidil (300 nmol/L)–induced dilations of denuded control arteries [C(-ENDO)] and arteries from diabetic animals (DM) (n=4 or 5). C indicates control arteries with intact endothelium.

View larger version (21K): [in this window] [in a new window]   Figure 10. Proposed scheme for the effects of STZ-induced diabetes mellitus on membrane potential, myogenic tone, and KATP channel opener sensitivity. Diabetes reduces the endothelial release of NO, leading to membrane potential depolarization of the vascular smooth muscle cells in the arterial wall. The membrane potential depolarization observed in arteries from diabetic animals would activate voltage-dependent Ca2+ channels,21,23,42 which increase Ca2+ entry and thus intracellular Ca,2+ leading to increased vasoconstriction. A decrease in basal NO release may alter the KATP channel opener responses directly and/or indirectly through the associated membrane potential depolarization (indicated by dashed lines). Reduced NO release may also constrict by mechanisms independent of changes in membrane potential (as indicated by a dashed line).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We have demonstrated that diabetes mellitus induced by STZ causes a membrane potential depolarization of 5 mV (at 60 mm Hg) and vasoconstriction. These findings can be explained by a loss in the tonic release of NO from the endothelium. The decrease in NO release or the membrane potential depolarization leads to a decrease in the apparent effectiveness of K_ATP channel openers, such as pinacidil and levcromakalim. Our results are summarized in the working hypothesis scheme shown in Fig 10.

We show for the first time that pressure-induced constriction ("myogenic tone") is enhanced in cerebral vessels from (STZ-induced) diabetic animals (Figs 1 and 2) in vitro. We did not detect changes in the passive properties of the cerebral arteries from diabetic animals compared with normal littermates (Fig 3). However, consistent with observations of others,^5–7 the response of arteries to the endothelium-dependent vasodilator acetylcholine, which stimulates release of NO from the endothelial cells, was greatly reduced in diabetic animals (Fig 4A). Diabetes did not appear to alter sensitivity to nitrovasodilators (Fig 4B), consistent with the observation of Mayhan.¹⁰ These results, taken together, are consistent with diabetes interrupting basal NO release from the endothelium without changing the properties of the vascular smooth muscle in this model of insulin-dependent diabetes.

The decreased sensitivity of pressurized cerebral arteries from diabetic rats to K_ATP channel openers (pinacidil and levcromakalim) that we observed is consistent with the previous observation of Mayhan and Faraci¹⁵ (1993) and Mayhan¹⁶ (1994) of decreased sensitivity of cerebral arteries to the K_ATP channel openers aprikalim and levcromakalim. We found that the difference in K_ATP channel opener sensitivity between normal and diabetic arteries could be abolished by removal of the endothelium (Figs 5 and 6) or by an inhibitor of NO synthase (LNNA) (Fig 7) (also see scheme in Fig 10). Furthermore, in support of the proposed scheme in Fig 10, we demonstrate that SNP can increase pinacidil responses in denuded control arteries and in arteries from diabetic animals (Fig 9B). In contrast, Mayhan and Faraci did not observe an effect of the NO synthase inhibitor L-NMMA at 1 µmol/L. The reason for this discrepancy in the results is unclear and may relate to the concentration of L-NMMA used.^35–37

Removal of the endothelium or inhibition of NO synthase by LNNA caused the smooth muscle membrane potential of pressurized arteries from normal rats to depolarize by 5 mV, which should contribute to the observed increase in "myogenic" constriction. In contrast, arteries from diabetic animals were 5 mV more depolarized than control arteries, and LNNA had no effect on the membrane potential of these arteries. These results suggest that control arteries release NO tonically, which causes a tonic membrane potential hyperpolarization of 5 mV, and in arteries from diabetic animals, this tonic release of NO and, hence, membrane potential hyperpolarization are lacking. In further support of this mechanism, the exogenous NO donor, SNP at 100 nmol/L, hyperpolarized arteries from diabetic animals by 6 mV (Figs 9A and 10).

Since myogenic tone is thought to be a major contributor to vascular resistance and, hence, blood pressure,²⁰ our observations are consistent with those of Huang et al³⁸ that mice lacking the gene for endothelial NO synthase are hypertensive. Membrane depolarization could decrease the apparent sensitivity of a pressurized artery to K_ATP channel openers by increasing the contribution of other ionic conductances (voltage-dependent Ca²⁺ channels, K_Ca channels, and voltage-dependent K⁺ channels¹⁹) to the membrane conductance. This would decrease the relative contribution of K_ATP channel conductance to the overall membrane conductance; thus, a greater increase in the open state probability of K_ATP channels would be required (hence a higher concentration of K_ATP channel openers) for a change in membrane potential.^22,23

Another possibility for the differences in K_ATP channel opener response in diabetes would be that NO increases the contribution of K_ATP channels to the membrane conductance or increases K_ATP channel opener affinity for the channel, even through voltage changes. However, GLIB (10 µmol/L) did not alter diameter and membrane potential of arteries from normal rats (Fig 8), suggesting that K_ATP channels, in the absence of K_ATP channel openers, do not seem to contribute significantly to the total membrane conductance under our in vitro conditions.

Another key question that remains to be explored is how the tonically released NO is hyperpolarizing the tissue. It has been shown that NO activates guanylyl cyclase, thereby increasing levels of cGMP.³⁵ Subsequent stimulation of PKG can activate K_Ca channels in the smooth muscle cells.³⁹ Activation of these K⁺ channels would cause membrane potential hyperpolarization, which would close voltage-dependent Ca²⁺ channels.²¹ This would lead to a decrease in intracellular Ca²⁺ and consequently to vasodilatation. In support of this, SNP has been shown to cause membrane potential hyperpolarization of pressurized cerebral (Fig 9A) and coronary arteries.⁴⁰ Other mechanisms may contribute to the membrane potential–dependent component of relaxation of arteries to NO, including the direct effect of NO on an ion channel or "cross-activation" of PKG or PKA, which can also activate K_Ca channels. In addition to the direct activation of K_Ca channels by either PKA or PKG, both these kinases could enhance K_Ca channel activity through increasing the amplitude or frequency of Ca²⁺-release events ("Ca²⁺ sparks") originating from the SR. These release events are thought to occur through ryanodine-sensitive Ca²⁺ channels in the subsarcolemmal SR. Ca²⁺ sparks appear to be major regulators of K_Ca channel activity in myogenic cerebral arteries.⁴¹ Other reported cGMP-mediated effects resulting in relaxation, such as inhibition of Ca²⁺ channels, enhanced Ca²⁺ extrusion, and, especially, enhanced uptake of Ca²⁺ into the SR, which could increase Ca²⁺-spark frequency and/or amplitude, may represent additional synergistic pathways by which NO relaxes arterial smooth muscle.

Conclusion
In summary, we conclude that insulin-dependent diabetes mellitus induced by STZ causes a membrane potential depolarization and vasoconstriction⁴² (see Fig 10). These effects may be due to an inhibition of tonic release of NO from the endothelium. The mechanism by which diabetes affects NO production remains to be determined. The decrease in NO release or the membrane potential depolarization leads to a decrease in the apparent effectiveness of K_ATP channel openers such as pinacidil and levcromakalim (Fig 10).

	Selected Abbreviations and Acronyms

 

GLIB = glibenclamide KATP channel = ATP-sensitive K+ channel KCa channel = Ca2+-activated K+ channel L-NMMA = NG-monomethyl-L-arginine acetate LNNA = NG-nitro-L-arginine PKA = cAMP-dependent protein kinase PKG = cGMP-dependent protein kinase SNP = sodium nitroprusside SR = sarcoplasmic reticulum STZ = streptozotocin

	Acknowledgments

 
This study was supported by the National Heart, Lung, and Blood Institute grants HL-44455, HL-51728, and HL-67647 and National Science Foundation grant DCB-9019563. This study was performed during the tenure of a fellowship of the American Heart Association, Vermont Affiliate, Inc, to Dr Knot. We would like to thank Dr Issy Laher for helpful discussions regarding the manuscript.

	Footnotes

 
¹ Both authors contributed equally to this study.

Received June 20, 1997; accepted September 19, 1997.

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