1 Department of Veterans Affairs and 2 Department of Internal Medicine and the Cardiovascular Center, University of Iowa, Iowa City, Iowa 52246
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ABSTRACT |
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Insulin and insulin-like
growth factor I (IGF-I) influence numerous metabolic and mitogenic
processes; these hormones also have vasoactive properties. This study
examined mechanisms involved in insulin- and IGF-I-induced dilation in
canine conduit and microvascular coronary segments. Tension of coronary
artery segments was measured after constriction with
PGF2. Internal diameter of coronary microvessels
(resting diameter = 112.6 ± 10.1 µm) was measured after
endothelin constriction. Vessels were incubated in control (Krebs)
solution and were treated with
N
-nitro-L-arginine
(L-NA), indomethacin, or K+ channel inhibitors.
After constriction, cumulative doses of insulin or IGF-I (0.1-100
ng/ml) were administered. In conduit arteries, insulin produced modest
maximal relaxation (32 ± 5%) compared with IGF-I (66 ± 12%). Vasodilation was attenuated by nitric oxide synthase (NOS) and
cyclooxygenase inhibition and was blocked with KCl constriction.
Coronary microvascular relaxation to insulin and IGF-I was not altered
by L-NA, indomethacin, tetraethylammonium chloride,
glibenclamide, charybdotoxin, and apamin; however, tetrabutylammonium chloride attenuated the response. In conclusion, insulin and IGF-I cause vasodilation in canine coronary conduit arteries and
microvessels. In conduit vessels, NOS/cyclooxygenase pathways are
involved in the vasodilation. In microvessels, relaxation to insulin
and IGF-I is not mediated by NOS/cyclooxygenase pathways but rather
through K+-dependent mechanisms.
diabetes mellitus; coronary circulation; coronary microcirculation; dogs; potassium channels
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INTRODUCTION |
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INSULIN AND insulin-like growth factor I (IGF-I) are structurally related peptides capable of stimulating a variety of metabolic and mitogenic processes. Receptors for both hormones are heterodimeric transmembrane tyrosine kinase receptors that, when stimulated, activate multiple intracellular pathways, resulting in altered metabolic and growth regulatory processes (9). IGF-I has been proposed as a novel therapy for patients with type 1 diabetes mellitus and for severely insulin-resistant patients with type 2 diabetes. Previous studies examining the vascular effects of insulin and IGF-I have yielded conflicting results. Some studies have shown that insulin or IGF-I enhanced tone or caused constriction (18, 24). Other studies have shown vasodilation that is either nitric oxide synthase (NOS) dependent (3, 6, 10, 17, 19, 22-24) or nitric oxide (NO) independent (11). The disparate effects may be due to differences in species or vascular beds studied. For example, Juncos and Ito (7) showed that insulin caused dilation of efferent but not afferent arterioles of the rabbit kidney, demonstrating regional variation, even within an organ.
No previous study has examined the vascular effects of insulin and IGF-I on conduit and microvascular arteries of the heart, an organ that is impacted upon by multiple processes in diabetes. In this report, we tested the hypothesis that insulin and IGF-I have different responses and mechanisms of action in coronary conduit vs. microvascular vessels. We further explored potential mechanisms responsible for the hormonally induced vasodilation.
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METHODS |
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All protocols were approved by the University of Iowa Animal Care and Use Committee and the Veterans Affairs Research Committee and conform to the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (NIH publication no. 85-23, revised 1985).
Animals and tissue. Sixty-five adult mongrel dogs of either sex (5-8 kg) were killed with an overdose of pentothal sodium (50 mg/kg). Hearts were removed and immediately placed in cold (4°C), oxygenated (20% O2-5% CO2-75% N2) Krebs bicarbonate buffer solution (see Solutions and drugs). Coronary conduit arteries and ventricular microvessels were isolated and cleaned of fat and connective tissue.
Isolated vascular rings. Canine coronary arteries were studied using a standard isometric ring technique (13, 14). Segments of the right coronary, left circumflex, or left anterior descending coronary arteries (1-2 mm in diameter) were dissected and cut into 5-mm ring segments. In some ring segments, endothelium was removed by pulling silk suture through the vessel. Vascular rings were mounted on two stainless steel wire stirrups passed through the vessel lumen. One stirrup was attached to a force transducer (Grass FT03), and the other was attached to a micrometer microdrive to allow the vessel to be stretched by small increments. Each vessel apparatus was placed in a 10-ml jacketed organ bath containing Krebs buffer equilibrated at 37°C and aerated with 20% O2-5% CO2-75% N2. Isometric contractions and relaxations were measured on a physiological recorder. Coronary rings were individually stretched to the maximum of the length-developed tension relationship by repeated test exposures to 75 mM KCl at increasing vessel tension.
The coronary artery segments were allowed to stabilize 30 min before concentration-response curves in either control solution (normal Krebs) or Krebs solution with NIsolated microvessels. A standard in vitro pressurized arteriole preparation was used to study coronary microvessels (13, 14). Ventricular microvessels (75-175 µm intraluminal diameter and ~1 mm in length) were carefully removed from the myocardium and cleaned with the aid of a dissecting microscope (Olympus SZ-6045 stereo zoom microscope). Each end of the microvessel was cannulated with a glass micropipette and secured with 10-0 ophthalmic suture. The cannulated pipettes were attached to a hydrostatic pressure reservoir (20 mmHg) under conditions of no flow. The organ chamber was placed on the stage of an inverted microscope (Olympus CK40). Attached to the microscope were a video camera, a video monitor, and a calibrated video caliper (Boeckler Video encoder). The organ chamber was connected to a rotary pump that continuously circulated oxygenated Krebs buffer warmed to 37°C. An image of the microvessel was displayed on the video monitor, and intraluminal diameters were measured by manually adjusting the video micrometer. The resolution of the system allowed measurement of very small (1-2 µm) changes in vessel diameter.
Microvessels were allowed to equilibrate for 30 min at a distending pressure of 20 mmHg. KCl (50 mM) was added to the bath to test constrictor capacity. Microvessels were incubated for 30 min in Krebs buffer alone (control) or in the presence of one or more of the following inhibitors: Indo (10 µM), L-NA (100 µM), glibenclamide (1 µM), tetraethylammonium chloride (TEA, 1 mM or 10 mM), tetrabutylammonium chloride (TBA, 1 mM), 4-aminopyridine (4-AP, 0.5 mM), or combinations of L-NA and Indo, glibenclamide and TEA, or charbydotoxin (50 nM) and apamin (500 µM). To mimic the effects of euglycemic and hyperglycemic conditions, responses to insulin and IGF-I were studied using low (5.5 mM) and high (22 mM) glucose concentrations. The standard solution used in this study contained 11 mM glucose. KCl constriction was used in some vessels to examine the role of K+ channels on insulin- and IGF-I-induced dilation. Both hypertonic (addition of KCl to the bath) and isotonic (equimolar reduction of NaCl) solutions were used. Endothelin-1 (0.40-8.0 nM) was used to constrict microvessels to 30-60% of their resting diameter. Cumulative concentration-response relationships were evaluated for insulin or IGF-I (0.1-100 ng/ml) by adding the drug directly to the organ bath. A single dose of SNP (10Solutions and drugs.
Krebs solution contained (in mM): 120.0 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 23.0 NaHCO3, 1.2 KH2PO4, 11 glucose, and 0.025 EDTA. Solutions
were aerated with 20% O2-5% CO2-75%
N2 and were warmed to 37°C with pH maintained at 7.4. Insulin was obtained from Eli Lilly (Indianapolis, IN) and was diluted
in 0.01 normal HCl. IGF-I was obtained from Intergen (Purchase, NY) and
was diluted in 0.05 M acetic acid. Insulin and IGF-I were stored at
20°C until ready to use. Endothelin-1 was purchased from Peninsula Laboratories (San Carlos, CA). All other inhibitors and vasoactive agents were purchased from Sigma Chemical (St. Louis, MO). All solutions and vasoactive agents were prepared fresh on the day of the experiment.
Statistical analysis.
Results are expressed as percent dilation, with 100% representing the
difference from the constricted value with endothelin, PGF2, or KCl to the resting value (tension or diameter). All concentration-response curves were evaluated for changes in maximal
responses and differences at each dose using ANOVA with repeated
measures and the Fisher least-significant difference correction for
multiple comparisons. Comparisons of percent vasodilation under
different treatment conditions were performed. Significance of
differences among mean values of resting conditions, maximal effect,
and EC50 values was assessed with a one-way ANOVA. Data are
expressed as means ± SE; n indicates the number of
animals. Differences with P < 0.05 were considered significant.
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RESULTS |
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Canine coronary conduit artery responses.
Resting tension of conduit artery rings was 2.8 ± 0.1 and
2.7 ± 0.1 g in insulin and IGF-I control groups,
respectively, and was not different among any of the treatment groups
[P = not significant (NS)]. Developed tension to
PGF2 was similar among groups [2.0 ± 0.2 and
2.6 ± 0.6 g in insulin and IGF-I control groups, respectively (P = NS)]. In canine coronary arteries
constricted with PGF2
, both insulin and IGF-I produced
relaxation (Fig. 1). The highest
concentration of IGF-I (100 ng/ml) induced 68 ± 8% relaxation
and was greater than the relaxation induced by 100 ng/ml insulin
(33 ± 4%). Insulin and IGF-I did not produce relaxation in
vessels that had endothelial cells removed (
1.4 ± 8.8%
dilation for insulin and 11.3 ± 6.1% dilation for IGF-I at 100 ng/ml, n = 4). Cyclooxygenase (Indo, 10 µM) and NOS
(L-NA, 100 µM) inhibition blocked the insulin-induced
dilation and modestly attenuated (46%) the IGF-I-induced dilation
(Fig. 1). When coronary rings were constricted with KCl (30-40
mM), relaxation to insulin and IGF-I was completely abolished (Fig. 1).
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Coronary microvascular responses.
Baseline diameter was 107 ± 17 and 102 ± 16 µm
(P = NS) for microvessels in the control groups for
insulin and IGF-I, respectively. Baseline diameters were not different
between the control and any of the treatment groups. Microvessels were
constricted with endothelin (0.4-8.0 nM) to 43 ± 4 and
49 ± 5% resting diameter in the control insulin and IGF-I
groups, respectively. Insulin and IGF-I produced concentration-related
relaxation in canine coronary microvessels. The vasodilator responses
to insulin and IGF-I were similar, with maximal relaxation of 69 ± 6% for insulin and 69 ± 7% for IGF-I (Fig.
2). Inhibition of NOS, cyclooxygenase, or
both pathways did not alter insulin- or IGF-I-induced vasodilation in
coronary microvessels (Fig. 3). Thus
NOS/cyclooxygenase pathways appear not to be necessary in
insulin/IGF-I-induced vasodilation in coronary microvessels, in
contrast to their role in the coronary conduit arteries.
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DISCUSSION |
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In this study, insulin and IGF-I produced vasodilation in both canine coronary conduit arteries and microvessels. However, the mechanisms responsible for vasodilation differed in the two coronary vascular beds. In coronary conduit arteries, insulin produced less relaxation than IGF-I. Also, in conduit arteries, vasodilation induced by both hormones was endothelium dependent, was attenuated by NOS and cyclooxygenase inhibition, and was completely blocked when KCl was used to constrict the vessels. In canine coronary microvessels, relaxation to insulin was similar to IGF-I-induced relaxation. However, in contrast to conduit arteries, this relaxation was not mediated by NO, cyclooxygenase, ATP-dependent K+ channels, or small or large KCa channels; rather, hyperpolarization, via some other K+ channel(s), appeared to be involved in the vasodilation.
Previous studies that focused on the vasoactive properties of insulin and IGF-I yielded conflicting results (7, 10, 11, 17, 18, 22, 24). Differences among these studies include the vascular bed studied, vessel size examined, and methodological diversity. Few studies examined the effects of insulin and IGF-I in the coronary circulation. Recently, Hasdai et al. (5) showed that insulin and IGF-I produced 28 and 25% relaxation, respectively, of endothelin-constricted porcine coronary conduit arteries. This relaxation was endothelium independent and involved smooth muscle cell K+ channels. We also saw modest relaxation to insulin (32%) in canine coronary arteries. However, in our study, IGF-I produced a greater amount of vasodilation than insulin (66 vs. 32%). The current study also found that insulin- and IGF-I-induced relaxation was endothelial dependent in canine conduit arteries. Hasdai et al. did not investigate microvascular responses to insulin and IGF-I in the porcine coronary circulation.
Selective differences in insulin- and IGF-I-induced vasodilation between conduit coronary arteries and coronary microvessels were present. In this study, L-NA and Indo attenuated the dilation to insulin and IGF-I in conduit arteries; however, the same combination of inhibitors did not alter microvascular dilation. McKay and Hester (10) have shown that insulin-induced vasodilation in hamster cremaster muscle is NO dependent in second-order but not in third- or fourth-order arterioles. Kersten el al. (8) and Nagao et al. (12) reported that smaller blood vessels exhibited enhanced potency to aprikalim, an agonist that produces vasorelaxation through activation of ATP-sensitive K+ channels, suggesting that small arteries are more sensitive to the actions of hyperpolarizing vasodilators than are large arteries.
Our results also show that K+ channels mediate some of the insulin- and IGF-I-induced vasodilation in both coronary conduit arteries and microvessels. High extracellular K+ concentrations blocked insulin- and IGF-I-induced dilation in both coronary conduit arteries and microvessels. In coronary microvessels, the nonspecific K+ channel inhibitor TBA attenuated the response to insulin and IGF-I. However, when studies were performed to block a specific class of K+ channels, using glibenclamide (1 µM), an ATP-sensitive K+ channel inhibitor, or TEA (1 mM), a KCa channel inhibitor, normal vasodilation was observed to insulin and IGF-I. Comparable relaxation was also observed in the presence of the combination of glibenclamide and TEA or at a higher concentration of TEA (10 mM).
There are several possible explanations for the K+ channel findings of our study. The inhibitors chosen in this study may not completely inhibit their respective classes of K+ channels (4, 16). A distinct type of K+ channel may be involved in insulin- and IGF-I-induced dilation that is not blocked by the inhibitors used in our study. Insulin and IGF-I may produce vasodilation through redundant pathways or diverging signal cascades, which in turn affect K+ channel activity in different ways.
Limitations of the study. Our study did not address the reactivity of coronary vessels from diabetic animals in response to insulin and IGF-I. Previous investigations have demonstrated that some vascular dysfunction associated with diabetes can be mimicked using hyperglycemic solutions (15, 20, 21). However, in this study, neither high nor physiological glucose concentrations altered insulin- or IGF-I-induced vasodilation in coronary microvessels. The use of euglycemic and hyperglycemic solutions to assess the potential effect of varying levels of glycemia on insulin and IGF-I may mimic the hyperglycemia observed in diabetes; however, it does not produce the potential adverse effects on the vasculature that can result from chronic hyperglycemia. Studies using vessels from diabetic animals could produce different results.
Systemic circulating levels of insulin are ~0.2 ng/ml in the fasting state, which increase to 2 ng/ml after a glucose load. In the portal circulation, the concentration of insulin can be 10 ng/ml or higher. In patients with insulin resistance, plasma levels of insulin can be >5 ng/ml. IGF-I is present in the circulation in microgram quantities. However, >90% of plasma IGF-I is present as a 150,000 complex that is thought to be confined to the vascular compartment. The precise mechanisms by which circulating IGF-I gets to tissue sites of action are uncertain. Further compounding the difficulty in determining the extravascular concentrations of IGF-I are the recent findings that most tissues can synthesize both IGF-I and one or more of the six high-affinity IGF binding proteins (1, 2). The concentration range in this study for insulin and IGF-I was from 0.1 to 100 ng/ml. Effective relaxation was observed in this range in both coronary conduit and microvessels. Although complete relaxation to insulin and IGF-I was not achieved in this range, higher concentrations of these hormones would likely represent nonphysiological concentrations of insulin and IGF-I. Thus the range of doses used in these studies encompassed both physiological and supraphysiological concentrations of insulin and IGF-I. In summary, insulin and IGF-I cause vasodilation in canine coronary conduit arteries and microvessels. In conduit vessels, endothelial-dependent NOS/cyclooxygenase pathways mediate insulin- and IGF-I-induced vasodilation. In coronary microvessels, relaxation to insulin and IGF-I is similar and is not mediated by NO/cyclooxygenase pathways, but rather by hyperpolarization through K+ channels. ![]() |
ACKNOWLEDGEMENTS |
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We thank Nam Yong Lee for technical assistance and Dr. Fausto Loberiza for statistical analysis.
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FOOTNOTES |
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This work was supported by National Institutes of Health National Research Service Award F32HL-09198 (to C. L. Oltman) and Grants RO1 HL-51308 (to K. C. Dellsperger), DK-25421-20 (to R. S. Bar), and DERC DK-25295-20 (R. S. Bar), the Veterans Affairs/JDF Diabetes Research Center (to D. D. Gutterman, R. S. Bar, and K. C. Dellsperger) and VA Merit Review (to D. D. Gutterman and R. S. Bar), and a Heartland Affiliate American Heart Association Grant-in-Aid (to K. C. Dellsperger).
Address for reprint requests and other correspondence: C. L. Oltman, Cardiovascular Research (151), VA Medical Center, Highway 6 West, Iowa City, IA 52246 (E-mail: coltman{at}blue.weeg.uiowa.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 1 November 1999; accepted in final form 10 February 2000.
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