(Hypertension. 1996;28:202-208.)
© 1996 American Heart Association, Inc.
Articles |
Role of Nitric Oxide, Adenosine, and ATP-Sensitive Potassium Channels in Insulin-Induced Vasodilation
the Department of Physiology and Biophysics, University of Mississippi Medical Center, Jackson.
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Abstract |
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The resistance of various tissues to the vasodilator and metabolic effects of insulin may be an important risk factor in the genesis of hypertension observed in several pathological states. Because of this, it is important to understand the mechanisms by which insulin causes vasodilation. Because insulin is known to raise metabolism, one mechanism by which insulin causes vasodilation could be through metabolic vasodilation. Recently, however, it has been suggested that the insulin-induced vasodilation is mediated by the release of endothelium-derived nitric oxide. Using a model of muscle microcirculation (hamster cremaster), we examined the interactions between insulin, nitric oxide, and tissue metabolism to understand the potential mechanisms by which insulin causes vasodilation. Topical application of insulin (200 µU/mL) to the cremaster resulted in significant increases in arteriolar diameter. Second-order arteriolar diameter increased from 69.6±6 to 79.8±5 µm and fourth-order arteriolar diameter from 11.3±1 to 15.1±2 µm (n=8). During nitric oxide synthase inhibition, topical application of insulin caused significant vasodilation in both second- and fourth-order arterioles. In contrast, both adenosine receptor antagonism and blockade of ATP-sensitive potassium channels prevented insulin-induced increases in arteriolar diameter. Our findings suggest a role for increased tissue metabolism, particularly the metabolite adenosine, in mediating insulin-induced vasodilation.
Key Words: arterioles • insulin • microcirculation • vasodilation
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Introduction |
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For the past two decades, investigators have searched for mechanisms underlying the genesis and maintenance of hypertension associated with a number of pathological conditions. In several hypertensive states, such as obesity and essential hypertension, there is a positive relationship between circulating levels of plasma insulin and diastolic pressure as well as a relative insensitivity to insulin-mediated glucose uptake. These observations have led some investigators to propose that hyperinsulinemia can result in hypertension.1 Several investigators have studied the potential link between hypertension and hyperinsulinemia using hyperinsulinemic euglycemic clamp models.2 3 4 5 6 7 8 Overall, these studies have shown that hyperinsulinemia does not result in hypertension per se. In fact, in lean insulin-sensitive subjects, both acute and chronic hyperinsulinemia increase cardiac output and decrease total peripheral resistance and mean arterial pressure. In other conditions, however, such as obesity and essential hypertension (insulin-resistant conditions), the ability of insulin to cause increments in cardiac output or decreased peripheral resistance is blunted or abolished.9 10 11 12 13 14 15 These findings suggest that in some conditions associated with hyperinsulinemia, hypertension may be exacerbated by the inability of insulin to cause vasodilation.
Acute and chronic elevations in plasma insulin levels with controlled blood glucose cause decreases in arterial pressure and vascular resistance. The decreases are thought to be the result of the action of insulin on the vasculature to cause vasodilation and thus increase blood flow through the tissues. The mechanisms that underlie the vasodilator capabilities of insulin are unclear but important to our understanding of the potential role of hyperinsulinemia as a risk factor in hypertension. Because of the known effects of insulin to increase tissue oxygen consumption, one logical mechanism by which insulin causes vasodilation is through metabolic vasodilation,16 potentially through the activation of K-ATP channels.17 Recently, however, two separate laboratories18 19 have provided evidence that acute increases in limb blood flow (leg and forearm) caused by hyperinsulinemia are mediated by endothelium-derived NO. In contrast, Hall et al20 have reported that the cardiovascular effects of chronic hyperinsulinemia (7 days) in lean dogs are not attenuated by NOS inhibition. Thus, the mechanisms underlying insulin-induced vasodilation have yet to be clearly established.
The primary extrahepatic effect of insulin is to stimulate glucose uptake, the majority of which occurs in skeletal muscle. In this study, we examined the effects of insulin on microvascular caliber in a well-established model of muscle circulation, the hamster cremaster muscle. This preparation allows us to visualize the intact circulation from the level of the resistance vessels to the level of the capillaries, thus allowing us to assess directly the effects of insulin on vascular diameters and, therefore, blood flow. To this end, we have tested the following hypotheses: (1) Insulin causes vasodilation in the hamster cremaster; (2) insulin-induced vasodilation is mediated by endothelium-derived NO; and (3) insulin-induced vasodilation is mediated through the metabolic intermediate adenosine.
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Methods |
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The experimental protocols for this study were approved by the Institutional Animal Care and Use Committee of the University of Mississippi Medical Center and were carried out according to both the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health and the guidelines of the Animal Welfare Act.
Animal Preparation
Male golden hamsters (110 to 175 g, Charles River Laboratories, Wilmington, Mass) were anesthetized with 0.27 mL of 60 mg/mL sodium pentobarbital solution IP (The Butler Co). Once anesthetized, hamsters were intubated and a catheter was placed in the left jugular vein for infusion of pentobarbital sodium in 0.9% saline solution (5 mg/mL at 0.01 mL/min) for maintenance of anesthesia. Deep esophageal temperature was maintained at 37° to 38°C by convective heating. The hamsters breathed 30% O2/70% N2 spontaneously to mimic blood gases typical of conscious animals.21 A total of 41 hamsters were used in this study.
The cremaster muscle was prepared for in vivo microscopy by a modification of the method of Baez.22 The cremaster was spread over a clear Lucite pedestal, and the edges were secured to a Sylgard (Dow Corning Corp) ring with insect pins. During the dissection and experimental period, the cremaster muscle was continuously superfused with PSS containing (mmol/L) NaCl 131.9, KCl 4.7, CaCl2 2.0, MgSO4 1.2, and NaHCO3 20 at pH 7.35. The solution was equilibrated with 5% CO2/95% N2 and heated to maintain a superfusate temperature of 34°C. Except where noted, all drugs and chemicals used were obtained from Sigma Chemical Co. Hamsters were allowed a 30-minute postsurgical recovery period before the experimental protocol was begun.
Experimental Measurements
The microcirculation of the cremaster was observed with a microscope (Leitz Laborlux 12FS) fitted with a x32 long-working distance objective (numerical aperture, 0.4). The microscope image of the muscle was televised with a closed-circuit video camera (66 series, Dage-MTI) and displayed on a monitor (Sony). Vessel diameter was measured with a Colorado Video 321 analyzer modified to function as a video micrometer. With the use of this device, two movable lines are positioned on the inside walls of the vessel, and a direct current voltage proportional to the line separation is recorded with a computerized data-collection system. The accuracy of this system is ±1 µm. For each experiment, all analog data were collected with a Gateway 486/33 computer equipped with a Metrabyte 12-bit analog-to-digital converter. The data were collected at 1 Hz and stored to disk for later analysis. In general, although we recorded arteriolar diameters at 5-minute intervals (see experimental protocols), we followed the diameters throughout the experimental period to ensure that we did not miss any transient changes in dilation.
Drug Preparation
Fifty microliters of 100 U/mL purified pork insulin (Novo Nordisk Pharmaceuticals) was diluted to 50 mL with sterile saline to obtain a working stock of 100 mU/mL. This 100 mU/mL insulin stock was diluted in 100 mL of PSS to obtain final concentrations of 10 and 200 µU/mL. The NOS inhibitor L-NAME (10 µmol/L) was made daily by dissolving 0.0054 g L-NAME in PSS. ACh stock (100 mmol/L) was made by dissolving 0.00182 g ACh in 100 mL sterile saline. One milliliter of this 100 mmol/L stock was diluted in 100 mL PSS to obtain a final concentration of 1 µmol/L ACh. Adenosine stock solution (10 mmol/L) was prepared by dissolving 1.33 g adenosine in 500 mL sterile saline. One hundred microliters and 1 mL of this adenosine stock were diluted in 100 mL PSS to yield final concentrations of 10 and 100 µmol/L, respectively. The adenosine receptor antagonist DPSPX (Research Biochemicals International) (25 mg) was dissolved in sterile saline to a concentration of 10 mmol/L. One milliliter of this DPSPX stock was then diluted in 1 L PSS to a final concentration of 10 µmol/L. Glibenclamide was dissolved in 50 µL dimethyl sulfoxide and 500 µL of 1 mol/L NaOH (JT Baker) and then diluted to 1 L with PSS. We have shown previously23 that the dimethyl sulfoxide/NaOH solution does not alter arteriolar diameter. Cromakalim (25 mg) was dissolved in 1 mL dimethyl sulfoxide and diluted to 87.3 mL with sterile saline for a stock solution of 1 mmol/L. One milliliter of this cromakalim stock was diluted in 100 mL PSS for a final concentration of 10 µmol/L. SNP (Elkins-Sinn) (50 mg) was dissolved in sterile saline to yield a 1 mmol/L stock solution. One hundred milliliters of this SNP stock was diluted to 100 mL in PSS to yield a final concentration of 10 µmol/L.
Experimental Protocols
Insulin-Induced Vasodilation
After the 30-minute recovery period, control vessel diameters were measured. The cremaster was then superfused with PSS containing 10 µU/mL insulin for 5 minutes, and vessel diameter was measured. The superfusate insulin concentration was increased to 200 µU/mL insulin, and vessel diameter was measured every 5 minutes for 15 minutes. In this protocol, the diameters of second-, third-, and fourth-order arterioles were observed. A total of eight hamsters were used in this protocol.
Role of NO in Insulin-Induced Vasodilation
In these experiments, the cremaster muscle was superfused with PSS containing 10 µmol/L L-NAME during the 30-minute recovery period and throughout the experimental period. After the recovery period, control diameters were measured, and then insulin was added to the superfusate to yield a concentration of 200 µU/mL insulin. Vessel diameters of second-, third-, and fourth-order arterioles were measured every 5 minutes for 15 minutes. A total of eight hamsters were used in this protocol.
An additional four hamsters were used for assessment of the blockade of NOS by L-NAME. In these hamsters, resting diameters were measured after the 30-minute recovery period. ACh was then added to the superfusate bath to yield a concentration of 1 µmol/L, and the dilator response was recorded. The superfusate was switched to one containing 10 µmol/L L-NAME, the cremaster was allowed to equilibrate with the L-NAME for 30 minutes, and diameters again were measured. The dilator response to 1 µmol/L ACh was then determined in the presence of 10 µmol/L L-NAME.
Role of Adenosine in Insulin-Induced Vasodilation
After the 30-minute recovery period, resting diameters were recorded. The arteriolar dilator responses to 10 and 100 µmol/L adenosine were obtained. The superfusate solution was then switched to one containing 10 µmol/L of the adenosine receptor antagonist DPSPX, and the tissue was allowed to equilibrate with the DPSPX for 1 hour. At the end of 1 hour, diameters were recorded followed by the response to 15 minutes of 200 µU/mL insulin in the presence of 10 µmol/L DPSPX. The ability of DPSPX to block adenosine vasodilation was confirmed by determination of the dilator response to adenosine (10 and 100 µmol/L) during DPSPX superfusion. A total of five hamsters were used for this protocol.
An additional six control experiments were performed to show that the 1-hour equilibration period did not affect the dilator response to insulin. This protocol was identical to the DPSPX protocol except that no DPSPX was added to the superfusate. In another five control experiments, the specificity of DPSPX for adenosine-induced vasodilation was determined. In these hamsters, the dilator responses to 10 µmol/L adenosine and 1 µmol/L SNP were measured. The cremaster was then superfused with 10 µmol/L DPSPX for 1 hour, and the dilator responses to adenosine and SNP again were determined in the presence of DPSPX.
Role of K-ATP Channels in Insulin-Induced Vasodilation
The microvascular response to adenosine has been shown to be mediated by activation of K-ATP channels. A role for K-ATP channels in the response to insulin was determined in the following protocol. After a 30-minute recovery period, control vessel diameters were obtained, and the vasodilator response to 1 µmol/L cromakalim (a K-ATP channel agonist) was determined. The superfusate was then switched to one containing 10 µmol/L glibenclamide (a K-ATP channel antagonist), and the tissue was allowed to equilibrate with the glibenclamide for 30 minutes. At the end of the 30 minutes of equilibration, diameters were measured again, and insulin was added to the bath. The effect of 200 µU/mL insulin in the presence of glibenclamide was assessed every 5 minutes for 15 minutes. To confirm that K-ATP channels were blocked, we determined the response to 1 µmol/L cromakalim in the presence of 10 µmol/L glibenclamide. A total of five hamsters were used in this protocol.
Statistical Analysis
Except where noted, the statistical significance for data was determined with repeated measures ANOVA, with Dunnett's test for determination of differences from control and the Tukey test for multiple comparisons. Values of P<.05 were considered significant.
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Results |
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Insulin-Induced Vasodilation
The effect of insulin on arteriolar diameter was determined in eight hamsters exposed to superfusate insulin concentrations of 10 and 200 µU/mL. After a 5-minute exposure to 10 µU/mL insulin, arteriolar diameters were unchanged from control diameters (Fig 1
). When the superfusate insulin concentration was increased to 200 µU/mL, arteriolar diameters were significantly increased after 15 minutes in both second- and fourth-order arterioles. Third-order arteriolar diameter appeared to be increased at the end of 15 minutes of 200 µU/mL insulin, but because of variability in the response (one vessel vasoconstricted) to insulin in this group of vessels, the increase was not statistically significant. In these experiments, the maximal diameters as determined with 100 µmol/L adenosine were 93±3 µm in second-, 51±6 in third-, and 22±3 in fourth-order arterioles.
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Mechanisms of Insulin-Induced Vasodilation
NOS Inhibition and Insulin-Induced Vasodilation
To determine whether the observed insulin-induced vasodilation was mediated by endothelium-derived NO, we superfused the cremaster with PSS containing 10 µmol/L L-NAME and determined arteriolar diameter responses to 200 µU/mL insulin (Fig 2). In the presence of L-NAME, insulin caused a significant increase in arteriolar diameter in both second- and fourth-order arterioles at 10 and 15 minutes (n=8 for second- and third-order and n=7 for fourth-order arterioles). The increases in diameter caused by insulin in the presence of L-NAME appeared to be attenuated compared with increases observed in the absence of L-NAME. The attenuated response was most notable in the second-order arterioles, but this difference was only significant at a value of P<.1 (ANCOVA). As in the previous experiment, one third-order arteriole vasoconstricted, and thus the increase in diameter measured in this group of vessels on the whole did not achieve statistical significance. In these experiments, the maximal diameters determined by 100 µmol/L adenosine were 96.2±5 µm for second-, 48.7±3 for third-, and 28.5±2 for fourth-order arterioles.
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In four hamsters, we determined the efficacy of NOS blockade by 10 µmol/L L-NAME (Table 1) and found that 1 µmol/L ACh caused significant dilation of second- and fourth-order arterioles. After the 30-minute equilibration with 10 µmol/L L-NAME, diameters were not changed from control diameters. Addition of 1 µmol/L ACh failed to cause arteriolar dilation in the presence of L-NAME.
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Role of Adenosine
We examined the role of the metabolic intermediate adenosine as a potential mediator of insulin-induced vasodilation. In five hamsters, we determined the effects of the adenosine receptor antagonist DPSPX on insulin-induced vasodilation. Initially, 10 µmol/L adenosine significantly increased microvessel diameter from 56.2±3.7 to 66.3±4.9 µm in second-order and from 14.8±1.1 to 20.7±1.9 µm in fourth-order arterioles. After the 1-hour equilibration with DPSPX, the dilator response to 200 µU/mL insulin was completely prevented (Fig 3), as was the response to 10 µmol/L adenosine. Third-order arterioles responded in a similar fashion (data not shown). In sham-treated hamsters (Fig 3) after 1 hour, a 15-minute superfusion with 200 µU/mL insulin caused significant increases in arteriolar diameter (from 68.0±3.8 to 75.8±4.6 µm in second-order and 13.3±1.6 to 18.2±2.2 µm in fourth-order arterioles). DPSPX did not prevent vasodilation through nonspecific effects, because 1 µmol/L SNP significantly increased arteriolar diameter both before and after treatment with 10 µmol/L DPSPX (Table 2).
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) or after 1 hour of PSS superfusion (
). In DPSPX-treated hamsters (n=5), resting diameters of second-order arterioles were 56.2±3.7 µm before DPSPX and 58.5±2.4 after 1 hour; fourth-order arteriolar resting diameter was 14.8±1.1 µm before DPSPX and 15.5±1.6 after 1 hour. In sham-treated hamsters (n=5), second-order resting diameter was 68.0±3.8 µm and 67.7±3.7 after 1 hour; fourth-order resting diameter was 13.3±1.6 µm and 12.4±0.9 after 1 hour. *Significantly different from resting diameter (P<.05); 
significantly different from initial adenosine response (P<.05).
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K-ATP Channels
Some researchers have shown the vasodilator effects of adenosine to be transduced via K-ATP channels. In addition, there are some reports that K-ATP channels are involved in part of the cellular hyperpolarization response to NO.24 To determine whether K-ATP channels were involved in insulin-induced vasodilation, we used the K-ATP antagonist glibenclamide. As can be seen in Fig 4, in the presence of glibenclamide (10 µmol/L), insulin did not cause vasodilation. In this protocol, the 30-minute superfusion with glibenclamide did not significantly alter resting diameters but completely prevented the vasodilator response to 1 µmol/L cromakalim. Similar responses were seen in third-order arterioles (data not shown). That glibenclamide did not alter the ability of arterioles to dilate has been recently shown in similar experiments from our laboratory in which glibenclamide attenuated the dilator response during functional hyperemia and in response to cromakalim but did not alter the dilator response to SNP.23
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) arterioles to insulin during K-ATP inhibition with glibenclamide (GLIB, 10 µmol/L). In the presence of 10 µmol/L glibenclamide plus 200 µU/mL insulin, arteriolar diameters were unchanged. *Significantly different from resting diameter (P<.05, n=5); |
Discussion |
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The results from this study show that insulin causes vasodilation in a model of muscle microcirculation, the hamster cremaster muscle. There was a tendency for the observed insulin-induced vasodilation to be attenuated by NOS inhibition, but overall, NOS inhibition did not abolish dilation. In contrast, the vasodilator effect of insulin was completely blocked by the adenosine receptor antagonist DPSPX and by the K-ATP channel blocker glibenclamide. These data support a role for increases in tissue metabolism (specifically, the metabolic vasodilator adenosine) as a mediator in insulin-induced vasodilation.
Numerous investigators have reported that both in whole animal experiments and during measurement of limb blood flow, hyperinsulinemic conditions increase cardiac output and blood flow and decrease vascular resistance.2 3 4 5 6 7 8 To determine whether the reported insulin-induced increments in blood flow are the direct result of vasodilation, we assessed directly the effects of insulin on arteriolar diameter in a model of muscle microcirculation, the hamster cremaster muscle. This muscle model allows observation of microvessels in the tissue with intact circulation and real-time measurement of changes in arteriolar diameter during acute exposures to superfusate insulin.
In general, the cremaster muscle has one feed arteriole, the first-order arterioles. Subsequent vessel branches are classified according to branching order, yielding an anatomically and physiologically distinct hierarchy of second-, third-, and fourth-order arterioles.25 26 Generally, second-order arterioles are associated with the control of blood flow to the tissue, and fourth-order arterioles regulate distribution of flow within the tissue.
In this study, the cremaster muscle was superfused with two physiological concentrations of insulin (10 and 200 µU/mL). The higher insulin dose caused significant vasodilation in both second- and fourth-order arterioles. Third-order arterioles in general dilated in response to the higher insulin concentration; however, in both the initial insulin experiments and those carried out in the presence of L-NAME, one arteriole vasoconstricted, and the group data on the whole failed to reach statistical significance. When these vessels are eliminated from statistical analysis, the dilation observed in third-order arterioles in response to 15 minutes of 200 mU/mL insulin was significant both during insulin superfusion and in the presence of insulin plus L-NAME (about a 20% increase in diameter in both groups; data not shown).
Overall, our present findings support the work of other investigators who in recent years have reported that physiological increases in insulin result in significant elevations in measures of dorsal hand vein, forearm, calf, and leg blood flows as well as in whole animal models of hyperinsulinemia over plasma insulin concentrations ranging from 50 to 250 µU/mL.2 6 7 8 Because we have observed similar effects of insulin in the present study, our study represents a valid model of insulin-induced vasodilation.
As indicated above, acute increments in plasma insulin levels lead to vasodilation under a variety of conditions. However, the mechanisms mediating the vasodilator actions of insulin have not been fully elucidated. Since insulin exerts potent metabolic effects and since some tissue metabolites are potent stimuli for vasodilation, it is likely that reported increases in blood flow and vasodilation in response to increased insulin are a secondary response to the action of insulin to increase tissue metabolism.16 Recently, other investigators have challenged this notion with evidence that insulin acts to increase blood flow via production of endothelium-derived NO. These studies reported that the NOS inhibitor N
-monomethyl-L-arginine (L-NMMA) attenuated or abolished hyperinsulinemic-induced increases in human leg18 and forearm19 blood flows. In these studies, however, it is unclear whether the attenuation by L-NMMA is the result of a direct interaction with the insulin response or whether it is the result of a vasoconstrictor action of L-NMMA.
NO and Insulin-Induced Vasodilation
We examined the role of endothelium-derived NO in insulin-induced vasodilation in the hamster cremaster using the NOS inhibitor L-NAME. The concentration of L-NAME used, 10 µmol/L, effectively blocked the dilator response to ACh (Table 1
). It is important to note in these experiments that although the vasodilator response to ACh was blocked, treatment with L-NAME had no effect on arteriolar diameter. Thus, unlike other studies,18 19 the vasodilator responses observed in the present study were not influenced by the confounding effects of L-NAME–induced vasoconstriction. Therefore, any insulin-induced changes in arteriolar diameter in the presence of L-NAME can be attributed to mechanisms independent of endothelium-derived NO synthesis. In the presence of L-NAME, insulin caused significant increases in the diameter of both second- and fourth-order arterioles. The response to insulin appeared blunted in second-order arterioles compared with the response observed in untreated hamsters (approximately 5 µm dilation in L-NAME–treated versus 10 µm in untreated hamsters). Although the attenuation of the insulin response was not significant at the .05 level, it is possible that a small portion of the observed insulin-induced vasodilation may be the result of NO production. However, the effect of L-NAME inhibition to limit insulin-induced vasodilation appeared to be limited to second-order arterioles.
That the effect of NOS inhibition is apparently influenced by vessel order may indicate a possible mechanism for the vasodilator action of insulin. There is a general consensus that the diameters of larger first- and second-order arterioles, which control blood flow to the tissue, are controlled both by metabolic mechanisms and by changes in pressure and flow. On the other hand, the diameters of smaller arterioles are considered to be controlled primarily by tissue metabolism.27 Our laboratory has shown previously that acute NOS inhibition by L-NAME attenuated the dilator response to muscle contraction of larger (first- and second-order) but not smaller (third-order) arterioles.28 From these observations, we suggested that the control of the diameter of smaller arterioles is tightly regulated by tissue metabolism and does not depend on endothelium-derived NO. Our present finding that insulin-induced vasodilation in metabolically sensitive third- and fourth-order arterioles is not affected by NOS inhibition is consistent with the notion that insulin-induced vasodilation can be mediated by increases in tissue metabolism.
Adenosine and Insulin-Induced Vasodilation
Increases in tissue metabolism lead to increased concentrations of metabolic by-products in the tissue. One of these products, adenosine, is a potent vasodilator. Consequently, adenosine has been suggested as a likely mediator of metabolic vasodilation. The cardiovascular effects of adenosine have been shown to be mediated by a number of different receptor subtypes.29 In general, the vasodilator effects of adenosine are thought to be mediated through the A2 receptor,29 but other in vitro studies have shown that adenosine causes hyperpolarization of vascular smooth muscle cells by binding to the A1 receptor and activating K+ channels.30 The activation of K+ channels causes cellular hyperpolarization and a decrease in intracellular Ca2+ concentration, leading to vasodilation.30 It is thought that the K+ channel mediating the cellular hyperpolarization by adenosine is the K-ATP channel.31 32 We investigated the role of insulin-induced, metabolically dependent increases in tissue adenosine production as a mediator of insulin-induced vasodilation. We examined this potential mechanism in two separate protocols: first in experiments in which the A1 and A2 receptors were blocked, and second in experiments in which K-ATP channels were blocked. When the cremaster muscle was treated with the adenosine receptor antagonist DPSPX, insulin-induced vasodilation was blocked (Fig 3). Blockade of K-ATP channels with glibenclamide (a K-ATP channel antagonist) also prevented insulin-induced vasodilation (Fig 4). These findings strongly support a role for an increase in tissue metabolism, particularly the metabolic vasodilator adenosine, as a mediator in insulin-induced vasodilation.
It has been suggested that the vasodilator effect of adenosine in isolated coronary arteries is mediated by activation of calcium-activated K+ (KCa) channels.33 Other researchers have shown in membrane preparations that KCa channels activated by cromakalim can be inhibited by 10 µmol/L glibenclamide,34 the concentration used in the present study. In contrast, Dart and Standen30 have shown in vascular smooth muscle that K+ current activated by adenosine was not blocked by charybdotoxin, a blocker of the KCa channel, but was blocked by 5 µmol/L glibenclamide. Thus, at this time, a role for KCa channels in adenosine-induced vasodilation (and therefore, insulin-induced vasodilation) is not clear.
Insulin-Induced Vasodilation, NO, and Adenosine
A common cellular pathway for adenosine and NO in vascular smooth muscle relaxation has been identified as the K-ATP channel.24 Our data support a role for both adenosine and K-ATP channels, and perhaps NO, in insulin-induced vasodilation. First, a portion of the effects of insulin on second-order arterioles appears sensitive to NOS inhibition, whereas the metabolically controlled third- and fourth-order arteriolar responses to insulin were not appreciably altered by NOS inhibition. Next, during adenosine receptor blockade with DPSPX, insulin, overall, did not increase microvascular diameter although there was a trend for second-order arteriolar diameter to be increased at the end of the insulin treatment. This small dilation could be the result of NO formation. Finally, glibenclamide, a K-ATP channel antagonist, prevented insulin-induced vasodilation without any apparent increase in arteriolar diameters of second-order arterioles during insulin treatment, suggesting that K-ATP channels are activated during increases in tissue metabolism caused by insulin.
In summary, our studies demonstrate that in the hamster cremaster muscle, as in many other tissues, insulin causes vasodilation. A portion of this dilation may be sensitive to NOS inhibition in a vessel order–specific manner. Finally, our findings suggest a role for the production of the metabolic vasodilator adenosine in response to insulin, which may have vasodilator effects through activation of the K-ATP channel.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This research was supported by grants HL-43089, HL-09262, and HL-51971 from the National Institutes of Health, Bethesda, Md, and an American Heart Association Grant-in-Aid. The authors would like to thank Daniel Boyd for excellent technical assistance.
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Footnotes |
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Reprint requests to Robert L. Hester, PhD, University Mississippi Medical Center, Department of Physiology and Biophysics, 2500 N State St, Jackson, MS 39216-4505.
A portion of this study was previously presented in abstract form (FASEB J. 1995;9:A112).
Received December 19, 1995; first decision January 29, 1996; accepted April 8, 1996.
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