Increased Myogenic Tone and Diminished Responsiveness to ATP-Sensitive K+ Channel Openers in Cerebral Arteries From Diabetic Rats
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+ (KATP) 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 KATP 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, NG-nitro-l-arginine (LNNA), in control arteries decreased the sensitivity to KATP 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 KATP 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 KATP channel openers.
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 KATP 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 KATP 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 KATP 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 KATP channel openers. These findings have implications for the regulation of arterial diameter by pressure, NO, and KATP channel openers during diabetes.
Materials and Methods
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.
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 MΩ. 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 (DR) values were obtained from passive (0 mmol/L Ca2+ PSS) arterial diameter measurements and were calculated using the following equation: DR=Dx/D0, where Dx 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.
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⇓).
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⇓).
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.
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 others9,32,33 and supports the hypothesis that endothelial cells are dysfunctional in diabetes.
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 KATP Channel Openers Are Reduced in Diabetes
Vasodilations of cerebral arterioles to the KATP channel openers aprikalim and levcromakalim are reduced in diabetes.15,16 We extended this observation by demonstrating that dilations to other types of KATP 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 KATP 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⇓).
Dilations to KATP 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 KATP 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 KATP 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 KATP channel opener sensitivity is due to reduced release of endothelium-derived NO affecting the arterial smooth muscle cells.
In addition, in the absence of KATP channel openers, KATP channels do not appear to contribute significantly to the regulation of myogenic tone, since a KATP 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 KATP 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 KATP channels.
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⇓).
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 KATP 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 KATP 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 KATP channel openers aprikalim and levcromakalim. We found that the difference in KATP 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 KATP channel openers by increasing the contribution of other ionic conductances (voltage-dependent Ca2+ channels, KCa channels, and voltage-dependent K+ channels19) to the membrane conductance. This would decrease the relative contribution of KATP channel conductance to the overall membrane conductance; thus, a greater increase in the open state probability of KATP channels would be required (hence a higher concentration of KATP channel openers) for a change in membrane potential.22,23
Another possibility for the differences in KATP channel opener response in diabetes would be that NO increases the contribution of KATP channels to the membrane conductance or increases KATP 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 KATP channels, in the absence of KATP 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 KCa 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 KCa channels. In addition to the direct activation of KCa channels by either PKA or PKG, both these kinases could enhance KCa 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 KCa 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.
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 KATP channel openers such as pinacidil and levcromakalim (Fig 10⇑).
Selected Abbreviations and Acronyms
|KATP channel||=||ATP-sensitive K+ channel|
|KCa channel||=||Ca2+-activated K+ channel|
|PKA||=||cAMP-dependent protein kinase|
|PKG||=||cGMP-dependent protein kinase|
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.
↵1 Both authors contributed equally to this study.
- Received June 20, 1997.
- Accepted September 19, 1997.
- © 1997 American Heart Association, Inc.
Garcia MJ, McNamara PM, Gordon T, Kannel WB. Diabetes as a cardiovascular risk factor. Diabetes. 1974;23:105–112.
Ruderman NB, Gupta S, Sussman I. An overview. In: Ruderman NB, Williamson JR, Brownlee ML, eds. Hyperglycemia, Diabetes, and Vascular Disease. New York, NY: Oxford Press; 1992:3–20.
Nahser PJ, Brown RE, Oskarsson H, Winniford MD, Rossen JD. Maximal coronary flow reserve and metabolic coronary vasodilation in patients with diabetes. Circulation. 1995;91:635–640.
Nitenberg A, Valensi P, Sachs R, Dali M, Aptecar E, Attali JR. Impairment of coronary vascular reserve and ACH induced coronary vasodilation in diabetic patients with angiographic normal arteries and normal left ventricular systolic function. Diabetes. 1993;42:1017–1025.
Fujii K, Heistad DD, Faraci FM. Effect of diabetes mellitus on flow mediated and endothelial dependent dilation of the basilar artery. Stroke. 1992;23:1494–1498.
Mayhan WG. Impairment of endothelium dependent dilation of cerebral arterioles during diabetes mellitus. Am J Physiol.. 1989;256:H621–H625.
Mayhan WG. Impairment of endothelium dependent dilation of the basilar artery during diabetes mellitus. Brain Res. 1991;580:297–302.
Lüscher TF. Endothelial derived contracting factors and hypertension. In: Ryan US, Rubanyi GM, eds. Endothelial Regulation of Vascular Tone. New York, NY: Marcel Dekker Inc; 1992:275–296.
Brody MJ, Dixon RL. Vascular reactivity in experimental diabetes. Circ Res. 1964;14:494–501.
Mayhan WG, Faraci FM. Responses of cerebral arterioles in diabetic rats to activation of ATP sensitive potassium channels. Am J Physiol. 1993;265:H152–H157.
Harder DR. Pressure-dependent membrane depolarization in cat middle cerebral artery. Circ Res. 1984;55:197–202.
Brayden JE, Nelson MT. Regulation of arterial tone by activation of calcium-dependent potassium channels. Science. 1992;256:532–535.
Knot HJ, Nelson MT. Regulation of membrane potential and diameter by voltage dependent potassium channels in rabbit myogenic cerebral arteries. Am J Physiol. 1995;269:H348–H355.
Meininger GA, Davis MJ. Cellular mechanisms involved in the vascular myogenic response. Am J Physiol. 1992;263:H647–H659.
Rubart M, Patlak JB, Nelson MT. Ca2+ currents in cerebral artery smooth muscle cells of rat at physiological Ca2+ concentrations. J Gen Physiol. 1996;107:459–472.
Nelson MT, Quayle JM. Physiological roles and properties of potassium channels in arterial smooth muscle. Am J Physiol. 1995;268:C799–C822.
Nelson MT, Patlak JB, Worley JF, Standen NB. Calcium channels, potassium channels, and voltage dependence of arterial smooth muscle cell tone. Am J Physiol. 1990;259:C3–C18.
Taguchi H, Heistad DD, Kitazono T, Faraci FM. ATP-sensitive K+ channels mediate dilation of cerebral arterioles during hypoxia. Circ Res. 1994;74:1005–1008.
Hong KW, Pyo KM, Lee WS, Yu SS, Rhim BY. Pharmacological evidence that calcitonin gene-related peptide is implicated in cerebral autoregulation. Am J Physiol. 1994;266:H11–H16.
Kitazono T, Heistad DD, Faraci FM. Role of ATP-sensitive K+ channels in CGRP-induced dilatation of basilar artery in vivo. Am J Physiol. 1993;265:H581–H585.
Kleppisch T, Nelson MT. ATP sensitive potassium currents in cerebral arterial smooth muscle: pharmacological and hormonal modulation. Am J Physiol. 1995;269:H1634–H1640.
Standen NB, Quayle JM, Davies NW, Brayden JE, Huang Y, Nelson MT. Hyperpolarizing vasodilators activate ATP-sensitive K+ channels in arterial smooth muscle. Science. 1989;245:177–180.
Brayden JE, Halpern W, Brann, LR. Biochemical, and mechanical properties of resistance arteries from normotensive and hypertensive rats. Hypertension. 1983;5:17–25.
Faraci FM. Role of endothelial derived relaxing factor in cerebral circulation. Am J Physiol. 1991;261:H1038–H1042.
Faraci FM. Role of nitric oxide in regulation of basilar artery tone in vivo. Am J Physiol. 1990;259:H1216–H1221.
Gold ME, Wood KS, Byrns RE, Fukuto J, Ignarro LJ. NG-methyl L-arginine causes endothelium dependent contraction and inhibition of cGMP formation in artery and vein. Proc Natl Acad Sci U S A. 1990;87:4430–4434.
Robertson BE, Schubert R, Hescheler J, Nelson MT. cGMP dependent protein kinase activates calcium activated potassium channels in vascular smooth muscle. Am J Physiol. 1993;265:C299–C303.
Wellman GC, Bonev AD, Nelson MT, Brayden JE. Gender differences in coronary artery diameter involve estrogen, nitric oxide and KCa channels. Circ Res. 1996;79:1024–1030.
Nelson MT, Cheng H, Rubart M, Santana LF, Bonev AD, Knot HJ, Lederer WJ. Relaxation of arterial smooth muscle by calcium sparks. Science. 1995;270:633–637.