Mechanisms underlying obesity-related vascular dysfunction are unclear. This study examined the effect of diet-induced obesity on expression and function of large conductance Ca2+-activated potassium channel (BKCa) in rat pressurized small resistance vessels with myogenic tone. Male Sprague-Dawley rats fed a cafeteria-style high fat diet (HFD; ∼30% energy from fat) for 16–20 wk were ∼30% heavier than controls fed standard chow (∼13% fat). Obesity did not alter BKCa α-subunit function or α-subunit protein or mRNA expression in vessels isolated from the cremaster muscle or middle-cerebral circulations. In contrast, BKCa β1-subunit protein expression and function were significantly reduced in cremaster muscle arterioles but increased in middle-cerebral arteries from obese animals. Immunohistochemistry showed α- and β1-subunits were present exclusively in the smooth muscle of both vessels. Cremaster muscle arterioles from obese animals showed significantly increased medial thickness, and media-to-lumen ratio and pressurized arterioles showed increased myogenic tone at 30 mmHg, but not at 50–120 mmHg. Myogenic tone was not affected by obesity in middle-cerebral arteries. The BKCa antagonist iberiotoxin constricted both cremaster muscle and middle-cerebral arterioles from control rats; this effect of iberiotoxin was abolished in cremaster muscle arteries only from obese rats. Diet-induced obesity has contrasting effects on BKCa function in different vascular beds, through differential effects on β1-subunit expression. However, these alterations in BKCa function had little effect on overall myogenic tone, suggesting that the mechanisms controlling myogenic tone can be altered and compensate for altered BKCa expression and function.
- arteriolar remodeling
- large conductance calcium-activated potassium channel
- myogenic tone
obesity is an independent risk factor for cardiovascular disease, atherosclerosis, dyslipidemia, and stroke (58), with increased peripheral vascular resistance being a common and consistent feature (31, 50). Increased vascular resistance in obesity may result, in part, from decreased function of the large conductance Ca2+-activated potassium channel (BKCa) in vascular smooth muscle. This channel plays a central role in the regulation of vascular tone, both as a regulator of vasoconstriction and a target of vasodilator agents (35, 43). BKCa have a high conductance of ∼240 pS (35), and hence a relatively low level of BKCa activity can exert a profound hyperpolarizing effect on muscle cell membrane potential, and thus excitability, reducing Ca2+ entry through voltage-sensitive mechanisms and causing relaxation. BKCa are composed of four pore-forming α-subunits and four regulatory β-subunits (64, 65). The β1-subunit is highly expressed in various smooth muscle cells, including in arteries (37), and acts to increase the Ca2+- and voltage sensitivity of the channel (65). BKCa are also activated by vasodilator agents including epoxyeicosatrienoic acids (EETs) derived from the action of cytochrome P-450 on arachidonic acid (8), nitric oxide (NO), and prostacyclin via cGMP and cAMP, respectively (57). BKCa may also be stimulated by vasoconstrictor stimuli, through the generation of local Ca2+-sparks involving activation of ryanodine receptors located on the sarcoplasmic reticulum (36, 40, 52). BKCa can also modulate responses to contractile stimuli without the generation of Ca2+ sparks (69).
Some studies have examined vascular BKCa function in various metabolic disease models. Impaired BKCa function in arteriolar smooth muscle was present in models of insulin resistance (14), diabetes (15, 47), and genetic models of obesity (23) and hypertension (3). However, few studies have examined BKCa function in a diet-induced obesity model that reflects the etiology of primary human diet-related obesity. Furthermore, the mechanism by which BKCa function was inhibited in these studies is unclear, with reactive oxygen species (6, 20, 23) or altered BKCa subunit expression (4, 44, 45) suggested to underlie changes in vascular function. Some recent studies have suggested metabolic disease may selectively reduce vascular BKCa β1-subunit function, reducing the channel sensitivity to changes in membrane potential or intracellular [Ca2+]. Cerebral artery BKCa from β1-subunit knockout mice have reduced Ca2+ sensitivity, with the animals having elevated mean arterial blood pressure (BP) and elevated pressure-induced cerebral myogenic tone (5). In models of diabetes and hypertension, reduced BKCa Ca2+ sensitivity in cerebral arteries was associated with decreased β1-subunit expression (2, 3, 15) and increased myogenic tone in retinal arteries (47).
The hypothesis of the current study was that diet-induced obesity alters BKCa function and regulation of myogenic tone in rat resistance arteries through altered expression of the BKCa β1-subunit. The study examined the effect of diet-induced obesity on vascular BKCa expression, function, and myogenic tone in two distinct vascular beds, cremaster (skeletal) muscle arterioles and middle-cerebral arteries. Pressure-induced myogenic constriction is an important component of overall arteriolar tone from both the skeletal muscle and cerebral circulations, and BKCa are a key regulator of myogenic tone in these vessels (41, 52). The present study utilized a well-characterized high fat, cafeteria-style dietary model of obesity. Animals subject to this diet for 16 wk display significant weight gain as well as mild systolic hypertension, hyperinsulinemia, and hyperleptinemia (29, 67).
MATERIALS AND METHODS
Animals, Diet, and Sample Collection
Male Sprague-Dawley rats (starting age 6 to 7 wk; 212 ± 1 g; n = 137) were fed either standard rat chow (Gordon's Specialty Stockfeeds) or a highly palatable cafeteria-style high-fat diet (HFD) and tap water ad libitum for 16–20 wk, as described previously (29). Protocols were approved by the University of New South Wales Animal Care and Ethics Committee.
The HFD consisted of foods such as pies, cakes, and biscuits in addition to standard chow. Body weight was measured weekly, and 24-h food intake (in kJ) was measured every 2 to 3 wk. At the end of the diet period rats were anesthetized with sodium thiopentone (100 mg/kg ip; Abbott). Blood samples were taken by cardiac puncture, and blood glucose was measured immediately (Accu-Chek Advantage; Roche Diagnostics Australia). Plasma samples were stored at −20°C for subsequent measurement of plasma insulin and leptin (Linco Research). Left retroperitoneal and testicular fat were removed and weighed.
Measurement of BP and Heart Rate
BP and heart rate was measured noninvasively with a tail-cuff volume pressure recording system (CODA 6; Kent Scientific Corporation) and CODA data acquisition software (V2; Kent Scientific). During week 1 of the diet period both control and HFD-fed rats (n = 12 of each) were allowed to acclimatize to the holders and tail-cuffs in three training sessions. Measurements were taken at weeks 1 and 16 of the diet period. For each animal 10–15 measurements were taken in a session. The CODA data acquisition software allowed continuous observation of volume pressure recordings in real-time. Measurements were only excluded if movement of the rat altered the waveform. For each rat 3–10 measurements were averaged for each session.
Pooled samples of first-order cremaster muscle arterioles or the main branches of middle-cerebral arteries were dissected and stored in liquid nitrogen from between 2 and 5 animals/n (n = 6 to 7 of each). The vessels were ground in liquid nitrogen using a mortar and pestle, and RNA was extracted using an RNeasy Mini kit (Qiagen). RNA was quantified using a spectrophotometer (Bio-Rad). Equal amounts of RNA samples were reverse-transcribed into cDNA using a QuantiTect reverse transcription kit (Qiagen). Quantitative RT-PCR was performed using a Mastercycler epgradientS realplex2 (Eppendorf). Primers were designed to amplify the BKCa α-subunit [forward primer 5′-CTCGAAGTGAAGCTGCCATGA, reverse primer 5′-ACTCCCGCTTGAGGTACTCGA; (47)], BKCa β1-subunit [forward primer 5′-ACCAATCTCTTCTGCACAGCAGC, reverse primer 5′-AGAGCTGTGACTGGCAGTTCCTT; (47)], and 18S rRNA [forward primer 5′-CCAGTAGCATATGCTTGTCTCAA, reverse primer 5′-CGACCAAAGGAACCATAACTGATT; (27); Sigma-Aldrich]. The reaction mixture contained 10 μl of 2× SYBR Green PCR Master Mix (Applied Biosystems), 4 μl of cDNA, 2 μl each of the relevant forward and reverse primers (final concentration, 500 nmol/l), and RNAse-free water added for a total volume of 20 μl. All reactions were performed in triplicate as follows: 10 min at 95°C followed by 40 cycles of 95°C for 15 s and 61°C for 1 min. Melting curves were performed for analysis of PCR product purity. Relative quantification analysis was performed using Realplex 2.0 software (Eppendorf).
Pooled segments of first-order cremaster muscle arterioles or the main branches of middle-cerebral arteries from between 3 and 9 animals/n (n = 3 to 4 of each) were dissected and stored in liquid nitrogen. The vessels were ground in liquid nitrogen using a mortar and pestle, resuspended in PBS (pH 7.4) containing complete protease inhibitor cocktail (Roche), and centrifuged (3,000 g, 4°C, 5 min). The supernatant was removed and placed on ice and the pellet was snap frozen in liquid nitrogen and processed again as described above. After the second spin the supernatants were pooled and centrifuged (25,000 g, 4°C, 1 h). The membrane-enriched pellet was carefully resuspended in PBS containing 0.1% Triton X-100 and protease inhibitor cocktail, aliquoted, snap frozen in liquid nitrogen, and stored at −80°C. Protein concentration of the samples was determined using the Bradford protein assay (Bio-Rad).
Aliquots of protein extracts (5 μg protein) were dissolved in lithium dodecyl sulfate (LDS) sample buffer of 0.5% LDS, 62.5 mmol/l Tris·HCl, 2.5% glycerol, 0.125 mmol/l EDTA (pH 8.5) for 10 min at 70°C. The samples were separated by electrophoresis in bis-Tris polyacrylamide gels using MES SDS running buffer and electroblotted onto polyvinylidene difluoride membranes overnight at 4°C (Invitrogen). After transfer, blots were thoroughly washed, blocked, probed with primary antibody to BKCa α-subunit (KCNMA1, 1:500; APC-107, Alomone, Israel); BKCa β1-subunit (KCNMB1, 1:2,000; to amino acids 118–132, which was a generous gift from Maria Garcia, Merck Pharmaceutical, New Jersey) and specific binding was visualized using alkaline phosphatase-conjugated secondary antibody [Rabbit IgG (Fc portion), Invitrogen; WB7106] and chemiluminescence, according to the manufacturer's instructions (Invitrogen). The blots were stripped and reprobed with actin antibody (1:1,000; Sigma A2066). The intensity of the band corresponding to each full-length protein was quantified by digital densitometry using ImageJ software (National Institutes of Health, Bethesda, MD). Relative intensity for each full-length band was determined by comparison with the intensity of actin staining.
To determine specificity, each antibody was incubated with its cognate peptide to block specific binding. Before use, peptide was added to antibody in a 1-to-1 ratio (wt/wt), mixed, and incubated at 37°C for 1 h, then overnight at 4°C. The blocked antibody was then used in Western blotting detection as described above. Antibody specificity was demonstrated previously (69).
Short segments of first-order arterioles from the cremaster muscle and the main branches of middle-cerebral arteries were dissected and pinned to a dish containing Sylgard (Dow Corning). Vessels were fixed in cold acetone for 10 min and washed with PBS. Vessels stained for BKCa α-subunit were preincubated for 2 h in a primary blocking buffer containing 1% BSA (Bovogen Biologicals) and 0.1% Triton X-100 in PBS. For BKCa β1-subunit staining the primary blocking buffer also contained 1% heat inactivated goat serum (1 h, 56°C). Vessels were incubated overnight at 4°C in antibodies against α-subunit (Alomone, APC107; 1:500) and β1-subunit (Merck anti-β1 to amino acids 118–132; 1:5,000), both diluted in primary blocking buffer. After washing in PBS (3 × 5 min) vessels were incubated in Alexa 633 goat-anti-rabbit (Invitrogen; 1:100) diluted in a secondary blocking buffer (0.01% Triton X-100 in PBS) for 2 h at room temperature in the dark. After vessels were washed in PBS (3 × 5 min), vessels were mounted using anti-fade mounting medium (DakoCytomation) and imaged in a confocal microscope (Olympus FV1000), using appropriate filters and uniform settings. Single section scans (0.1–0.2 μm) were made to construct the confocal stacks shown.
Isolated cremaster muscle arteriole and middle-cerebral artery preparation.
Cremaster muscles were dissected and transferred to a chilled (4°C) dissecting dish containing buffer (in mmol/l) of 3 MOPS, 145 NaCl, 5 KCl, 2.5 CaCl2, 1 MgSO4, 1 NaH2PO4, 0.02 EDTA, 2 pyruvate, 5 glucose, and 1% BSA. The brain was dissected and transferred to a dissecting dish containing modified Krebs buffer (in mmol/l) of 111 NaCl, 25.7 NaHCO3, 4.9 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 11.5 glucose, and 10 HEPES. A segment of the first-order cremaster muscle arteriole or the main branch of the middle-cerebral artery was isolated and transferred to a tissue bath. The tissue bath contained modified Krebs buffer solution bubbled with 5% CO2-95% N2. This gas mixture is used as resistance vessels are routinely exposed to and function at O2 tensions substantially lower than large arteries (16, 33). The isolated vessel was cannulated onto glass micropipettes and secured with 10-0 sutures (Alcon Laboratories).
Responses to BKCa agonists.
In isolated, pressurized (70 mmHg) cremaster muscle arterioles or middle-cerebral arteries (80 mmHg) cumulative concentration-response curves to the BKCa agonists 1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one (NS1619; 1–100 μmol/l), pimaric acid (PiMA; 0.1–30 μmol/l; Alomone Labs, Israel), and tamoxifen (0.01–100 μmol/l) were performed alone and in the presence of the BKCa inhibitor iberiotoxin (IBTX; 0.1 μmol/l).
After development of spontaneous tone in cremaster muscle arterioles or middle-cerebral arteries (at 70 or 80 mmHg, respectively) the pressure-diameter relationship was measured over the pressure range of 30–120 mmHg in the absence and presence of IBTX (0.1 μmol/l). Pressure changes were randomized, and the vessels were allowed to equilibrate for 5 min at each pressure. At the conclusion of all experiments the passive pressure-diameter relationship was measured in a nominally Ca2+-free Krebs solution (no added CaCl2 and 2 mmol/l EGTA).
Tissue preparation for electron microscopy was as described previously (60). In brief, first-order cremaster muscle arterioles from control and obese rats (n = 10, each from a different animal) were dissected from perfusion fixed rats [1% paraformaldehyde, 3% glutaraldehyde in 0.1 mmol/l sodium cacodylate buffer, with 10 mmol/l betaine (pH 7.4)], embedded, sectioned, and imaged at ×10–40 k on a Phillips 7100 transmission electron microscope at 16 megapixel (MP) resolution (camera from Scientific Instruments and Applications, Duluth). Quantitative measurements of wall properties were made from vessel cross sections cut 90° perpendicular to the longitudinal vessel axis from ultrastructural montages taken at ×1.5–2.5 k at 16 MP resolution. CellR software (Olympus) was used for gross quantitative measurements.
Results are expressed as means ± SE. Diameter is expressed as a percentage of the passive diameter observed in nominally Ca2+-free Krebs solution. EC50 was calculated by nonlinear regression of individual concentration-response curves (GraphPad Prism v5). Data were analyzed using a two-way ANOVA followed by t-test for individual groups of data or t-test alone where warranted (GraphPad Prism v5).
Drugs and Chemicals
All chemicals were from Sigma-Aldrich (St. Louis, MO), except where stated. NS1619 and PiMA were dissolved in DMSO and ethanol, respectively, to make a 10 mmol/l stock solution and stored at −20°C. Vehicle solutions had no effect on vessel responses. IBTX was dissolved in distilled water to make a 10 μmol/l stock solution and stored at −20°C. Tamoxifen 10 mmol/l stock solutions were prepared fresh daily in ethanol. Both PiMA and tamoxifen 1 mmol/l stock solutions contained 37% ethanol to prevent precipitation. All stock solutions were diluted in Krebs buffer to achieve the final concentration.
Effect of HFD
During the diet period the daily energy intake of animals maintained on the HFD was significantly increased compared with control (P < 0.05; Table 1). The HFD consisted of 32.1 ± 1.0% energy from fat, and the control diet consisted of 13.7% (no SE since only 1 food source). At the end of the diet period the obese animals on the HFD had increased body weight and fat mass (P < 0.05). Nonfasting blood glucose, plasma insulin, and leptin were also increased (P < 0.05).
Effect of HFD on BP and Heart Rate
During the first week of the diet period there was no difference between systolic/diastolic BP or heart rate between control (121 ± 6/90 ± 5 mmHg, 408 ± 8 beats/min) and HFD-fed rats (127 ± 5/92 ± 5 mmHg, 411 ± 6 beats/min, P > 0.05; unpaired t-test, n = 11 to 12 of each). At the end of week 16 systolic BP, but not diastolic BP or heart rate, was significantly elevated in HFD-fed (obese, 158 ± 4/110 ± 3 mmHg, 425 ± 15 beats/min) rats compared with control (144 ± 2/100 ± 3 mmHg, 433 ± 11 beats/min, P < 0.05; two-way ANOVA, n = 11 to 12 of each).
Effect of Diet-induced Obesity on Cremaster Muscle Arteriole and Middle-cerebral Artery Diameter
At 70 mmHg, the diameter of cremaster muscle arterioles from obese rats was not significantly different from control (80.4 ± 2.5 μm vs. 81.3 ± 1.9 μm, respectively, P > 0.05; unpaired t-test, n = 41–43 of each). The passive (maximum) diameter of cremaster muscle arterioles from obese rats was significantly increased compared with control (177.5 ± 3.1 μm vs. 168.3 ± 2.4 μm, respectively, P < 0.05; unpaired t-test). Myogenic tone expressed as a percentage of passive diameter was not significantly different between the two groups, 45.5 ± 1.3% in obese and 48.3 ± 1.0% in control (P > 0.05; unpaired t-test).
In middle-cerebral arteries maintained at 80 mmHg, the diameter of arteries from obese rats was not significantly different from control (175.6 ± 6.1 μm vs. 174.0 ± 6.2 μm, respectively, P > 0.05; unpaired t-test, n = 26 of each). Passive (maximum) diameter of middle-cerebral arteries from obese rats was similar to control (288.3 ± 5.8 μm vs. 283.7 ± 6.7 μm respectively, P > 0.05; unpaired t-test). Myogenic tone expressed as a percentage of passive diameter was not significantly different between the two groups, 61.2 ± 2.1% in obese and 61.7 ± 2.0% in control (P > 0.05; unpaired t-test).
Effect of Diet-induced Obesity on mRNA Expression of BKCa α- and β1-subunits in Cremaster Muscle Arterioles and Middle-cerebral Arteries
Real time-PCR showed that mRNA expression of BKCa α- (Fig. 1, A and D, respectively) or β1-subunits (Fig. 1, B and E, respectively) relative to 18S rRNA, in both cremaster muscle arterioles (Fig. 1, A–C) and middle-cerebral arteries (Fig. 1, D–F) from obese rats, was not significantly different from control. There was also no difference in the ratio of β1- to α-subunit in either vessel from control or obese rats (Fig. 1, C and F). Melt curves demonstrated the presence of a single consistent product for each of the subunits. For details, see Supplemental Fig. S1.
Effect of Diet-induced Obesity on Protein Expression of BKCa α- and β1-subunits in Cremaster Muscle Arterioles and Middle-cerebral Arteries
Western blotting of cremaster muscle arteriole and middle-cerebral artery proteins showed that BKCa α-subunit expression was similar in obese and control rats (relative to actin; Fig. 2, A and D and F and I, respectively). In obese rats there was a significant reduction in β1-subunit expression in cremaster muscle arterioles (P < 0.05; Fig. 2, B and E). In middle-cerebral arteries from obese rats, however, expression of β1 was increased (P < 0.05; Fig. 2, G and J). Furthermore, the ratio of β1- to α-subunit expression was decreased in cremaster muscle arteriole and increased in middle-cerebral arteries from obese rats (P < 0.05; Fig. 2, C and H, respectively). In control rats, the ratio of β1- to α-subunit expression was significantly lower in middle-cerebral arteries compared with cremaster muscle arterioles (0.25 ± 0.07 vs. 0.51 ± 0.01, respectively, P < 0.05; unpaired t-test). Peptide block of the primary antibody abolished staining of bands corresponding to the predicted molecular weight of α- and β1-subunit proteins (39, 46). For detail, see Supplemental Figs. S2, S3, and S4.
BKCa α- and β1-subunit Detection by Immunohistochemistry in Cremaster Muscle Arterioles and Middle-cerebral Arteries
BKCa α- and β1-subunits were detected exclusively in the smooth muscle of cremaster muscle arterioles (Fig. 3, A–F) and middle-cerebral arteries (Fig. 3, G–L), being absent from the endothelium. In cremaster muscle and middle-cerebral vessels from control and obese animals, the α- and β1-subunits were present on the cell membrane, and also in the cytoplasm, where distribution appeared to reflect that of the sarcoplasmic reticulum. Dual α- and β1-subunit labeling could not be performed since characterized primary antibodies came from the same host. Peptide block of the primary antibody abolished staining.
Effect of Diet-induced Obesity on Responses to BKCa Agonists in Cremaster Muscle Arterioles And Middle-cerebral Arteries
The BKCa α-subunit-selective activators NS1619 (0.1–300 μmol/l; Fig. 4, A and D) and PiMA (0.1–30 μmol/l; Fig. 4, B and E) and the β1-subunit selective activator tamoxifen (0.01–100 μmol/l; Fig. 4, C and F) caused concentration-dependent dilation of pressurized cremaster muscle arterioles (Fig. 4, A–C) and middle-cerebral arteries (Fig. 4, D–F). Responses of either vessel from both control and obese rats to NS1619 or PiMA were not significantly different (Fig. 4, A, B, D, and E). However, tamoxifen was a less potent vasodilator in cremaster muscle arterioles from obese animals (pEC50; control, 5.8 ± 0.2 vs. obese, 5.0 ± 0.1; P < 0.05, unpaired t-test; n = 6–10 of each; Fig. 4C). In contrast, in middle-cerebral arteries, the vasodilatory potency of tamoxifen was increased in obese rats, compared with control (pEC50; control, 5.1 ± 0.2 vs. obese, 6.0 ± 0.2; P < 0.05, unpaired t-test; n = 9 of each; Fig. 4F). In control animals, tamoxifen was a less potent dilator in middle-cerebral arteries compared with cremaster muscle arterioles (P < 0.05, unpaired t-test; n = 6–9 of each), whereas the opposite was true in the respective vessels from obese animals. IBTX significantly inhibited, but did not abolish, dilation to NS1619 and PiMA in both cremaster muscle arterioles and middle-cerebral arteries from both control and obese rats (Fig. 4, A, B, D, and E). Dilation to tamoxifen was abolished by IBTX in both vessels from control and obese rats (Fig. 4, C and F).
Effect of Diet-induced Obesity on Myogenic Tone in Cremaster Muscle Arterioles and Middle-cerebral Arteries
At low pressure (30 mmHg) myogenic tone was increased in cremaster muscle arterioles from obese compared with control rats (Fig. 5C). At 30 mmHg the diameter of obese arterioles was 68.3 ± 5.7% (of passive diameter, 124.5 ± 4.5 μm) compared with 85.9 ± 3.9% (passive 106.1 ± 2.3 μm) in control (P < 0.05). This increase in tone was not evident at higher pressures (50–120 mmHg). The passive (maximum) diameter of cremaster muscle arterioles from both control and obese rats was similar at all pressures except 30 mmHg, where passive diameter was greater in arterioles from the obese rats (above and Fig. 5D). IBTX significantly constricted cremaster muscle arterioles from control rats across the entire pressure range (30–120 mmHg), whereas in arterioles from obese rats IBTX had no significant effect on diameter, at any pressure (Fig. 5A).
Myogenic tone of middle-cerebral arteries from both control and obese rats was similar across the pressure range tested (30–120 mmHg; Fig. 5B). IBTX constricted middle-cerebral arteries from control rats across the entire pressure range, and this response was not altered in arteries from obese rats (Fig. 5B). The passive (maximum) diameter of middle-cerebral arteries from both control and obese rats was similar across the pressure range (Fig. 5E).
Effect of Diet-induced Obesity on the Ultrastructural Characteristics of Cremaster Muscle Arterioles
Increased active tone and passive diameter in cremaster muscle arterioles from obese rats, at low pressures, suggested some remodeling of the arteriolar wall. This was investigated further in perfusion fixed arterioles. In these vessels lumen diameter was not significantly different between arterioles from control and obese rats (Fig. 6; Table 2). The number of smooth muscle cell layers, medial thickness, and media-to-lumen ratio were significantly larger in cremaster muscle arterioles from obese rats (Fig. 6; Table 2). For detail, see Supplemental Table S1.
Consistent with obesity in humans, rats fed a cafeteria-style HFD had significant weight gain and increased fat mass. In addition systolic BP, nonfasting blood glucose, plasma insulin, and leptin were significantly elevated. Hence the changes in BKCa function observed in this study may better reflect vascular changes in humans compared with genetic models. There are multiple factors that may contribute to obesity-induced vascular dysfunction. Increased body weight and fat mass has been shown to correlate to hyperleptinemia in both humans and animal models of obesity (29, 50, 67). Increased sympathetic nervous system activity and thus BP is characteristic of obesity-associated hypertension in human and animal models, the etiology of which appears to be multifactorial involving insulin, leptin, and other hormonal factors (42, 50).
Inhibition of BKCa contributes to increased vascular resistance and BP in some models of obesity, hypertension, and diabetes (15, 23). The mechanisms underlying BKCa inhibition are not clear, since disparate results are found in different models, decreased (3, 15, 47), increased (4), or no change (6) in β1-subunit expression. In the same manner, BKCa function has also been shown to be decreased (4, 23) or increased (45). In the present study we aimed at a more expansive view by examining the effect of a dietary, rather than genetic, model of obesity on BKCa expression and function in small resistance vessels from different vascular beds. BKCa β1-subunit protein expression and BKCa function was reduced in cremaster muscle arterioles, but not middle-cerebral arteries, from obese rats. Indeed, β1-subunit protein expression and function (the latter assessed by tamoxifen sensitivity) was increased in middle-cerebral arteries from obese rats. Expression or function of the α-subunit was not altered in either vessel from obese rats. Consistent with previous studies (24), immunohistochemistry showed α- and β1-subunits were present exclusively in the smooth muscle of both vessels from both control and obese rats. It is possible that the antibodies used stained mitochondrial BKCa, which have been identified in the heart and brain (7).
The changes in BKCa β1-subunit protein expression in vessels from obese rats were not reflected at the mRNA level, suggesting that consequent changes in β1 expression and function arise at the post-transcriptional level. Neither BKCa β1-subunit mRNA expression nor the ratio of β1- to α-subunit mRNA was altered in either vessel from obese rats. These observations suggest that the differences in the quantity of β1-subunit protein in both vessels in obese animals are more likely due to changes in β1 protein metabolism rather than a change in overall β1 protein translation. The observations on mRNA levels are in contrast with those in the streptozotocin-induced type 1 diabetic rat, where both BKCa β1-subunit mRNA and protein expression (the latter measured by immunohistochemistry rather than Western blotting) were significantly reduced in retinal arterioles (47). Streptozotocin treatment appears to have a more severe impact on BKCa β1-subunit expression and function.
The ability of BKCa to modulate myogenic tone in cremaster muscle arterioles from obese rats was abolished, suggesting the β1-subunit is crucial to activation of BKCa by pressure-induced constriction. However, these alterations in BKCa function had little effect on overall myogenic tone, despite a small increase in tone at 30 mmHg, which was most likely due to vessel wall remodeling and reduced distensibility, rather than altered BKCa function, since passive (maximum) diameter at this pressure was also increased in obesity. In middle-cerebral arteries BKCa β1-subunit protein expression was increased in obesity, although there was no apparent increase in the role of BKCa in modulating myogenic tone or any change in overall tone. Consistent with previous studies in the cerebral circulation at physiological pressures, the myogenic response curve of middle-cerebral arteries was relatively flat across the pressure range tested (40, 54, 70). These observations suggest that the effects of diet-induced obesity on BKCa expression and function are heterogeneous between vessels from different beds. Furthermore, the mechanisms controlling overall myogenic tone can be altered and compensate for a loss or increase in BKCa expression and function. The mechanisms by which pressure-induced myogenic tone is produced and maintained are complex and not fully understood (30). Other mechanisms that may be altered in obesity to regulate myogenic tone include increased L-type Ca2+ channel activity (4) and altered activity of other K+ channels such as those of the voltage-activated type (KV; 59).
Changes in BKCa β-subunit expression also alter IBTX pharmacology. Dworetzky et al. (17) showed that BKCa-mediated currents in Xenopus oocytes expressing both α- and β-subunits were approximately tenfold less sensitive to inhibition by IBTX than BKCa currents in oocytes expressing the α-subunit only. Such observations do not correlate with effects observed in the present study, in that reduced β1-subunit expression also reduced the functional effects of IBTX. In this case, it seems more likely that reduced β1-expression reduced the ability of the pressure-stimulus to activate the channel. The effects of other BKCa β-subunits on IBTX binding affinity are well established (48), but these subunits are not expressed to a significant extent in vascular smooth muscle. Furthermore, given that reported changes in IBTX affinity for BKCa resulting from altered expression of any β-subunit (including β1) are usually in the picomolar to low-nanomolar range (17, 25, 48) we are confident that the concentration of IBTX used in our study (100 nM) maximally inhibited BKCa channels regardless of the level of β1-subunit expression.
In contrast with the observations of the present study, increased myogenic tone was associated with the loss of the β1-subunit in cerebral arteries from streptozotocin-induced type 1 diabetic rats (15) and BKCa β1-subunit knockout mice (5). It is possible that the reduction in β1-subunit protein expression observed in cremaster muscle arterioles in our obesity model, despite being sufficient to abolish the effects of IBTX, was not severe enough to affect myogenic tone. It is worth noting that, apart from one study in streptozotocin-induced diabetes (15), few investigations in this area have actually measured BKCa β1-protein expression. This may be due to the difficulty of working with small vessels, where it is necessary to pool tissue from several animals. It should also be mentioned that in both diabetic Zucker (6) and streptozotocin-treated (15, 47) rats the extent of hyperglycemia and hyperinsulinemia was much greater than in our dietary obese model, and these animals did not experience weight gain or hypertension compared with control animals. Furthermore, there is also evidence of increased frequency or amplitude of Ca2+ sparks; or altered coupling between Ca2+ sparks and BKCa in vascular smooth muscle cells from diabetic models (15, 47, 49). Of interest, in a recent study Ca2+ sparks could not be detected in smooth muscle from rat cremaster muscle arterioles (69). Nevertheless, the reduction in the β1-subunit in the present study was functionally significant, as shown by the inability of IBTX to constrict these vessels and the reduced response of intact arterioles to the BKCa β1-selective activator tamoxifen. Tamoxifen and the related compound 17β-estradiol are established, selective activators of the BKCa β1-subunit (11, 12, 13), and in the present study tamoxifen-induced dilation was abolished by IBTX in both vessels, suggesting this vasodilation was mediated exclusively by BKCa. In middle-cerebral arteries dilation to tamoxifen was relatively small and, combined with the relatively large constriction induced by IBTX, suggests a high level of BKCa activity, consistent with previous studies demonstrating the importance of BKCa in the cerebral circulation (52).
The present study found BKCa α-subunit mRNA and protein expression and function was not altered in either cremaster muscle arterioles or middle-cerebral arteries from obese rats. The different appearance of the smeared BKCa α-band (100–110 kDa) in cremaster muscle arterioles compared with middle-cerebral arteries is most likely due to α-subunit splice variants and/or phosphorylation sites (68). There was no evidence of reduced α-subunit expression or functional inhibition in either vessel from diet-induced obese rats, as established by selective α-subunit activators NS1619 (28) and PiMA (34) in the present study. IBTX abolished dilation induced by either NS1619 or PiMA over most of the active concentration range of these agents, in vessels from both control and obese rats, suggesting these effects were mediated solely by BKCa. It is well known that high concentrations of NS1619 also inhibit KV and L-type Ca2+ channels (18, 32), reflected in the present study by the inability of IBTX to inhibit the vasodilation caused by supramaximal concentrations of NS1619 (>30 μM).
Increased myogenic tone and passive diameter, at low pressures in cremaster muscle arterioles from obese rats, indicated remodeling. Electron microscopy showed that both medial thickness and media-to-lumen ratio were significantly increased in cremaster muscle arterioles from obese rats, which differs from the morphological changes described in studies using the obese Zucker rat (22, 63). In contrast with the diet-induced obesity model, reduced lumen diameter and atrophy of the vascular wall was observed in gracilis arteries from OZR, although in middle-cerebral arteries no remodeling was observed (63). In a separate study, cerebral arteries from OZR had increased myogenic tone and inward remodeling (53). Inward, eutrophic remodeling has been observed in cremaster muscle arterioles in one-kidney one-clip hypertension (55). In cerebral arteries from HFD-fed rats reduced lumen diameter, increased wall thickness and wall-to-lumen ratio were observed (10). There was no functional evidence of remodeling in middle-cerebral arteries from our diet-induced obese rats. Collectively, these data suggest that there is considerable heterogeneity in remodeling that is vascular bed and model specific.
The obesity-related factor(s) that caused the changes in BKCa β1-expression and BKCa function are difficult to identify. Of the factors measured (leptin, insulin, and blood glucose), insulin has been shown to increase BKCa expression and function in the plasma membrane of mouse podocytes, whereas high concentrations of glucose had the opposite effect (38). In contrast with the current findings, BKCa function was inhibited in cerebral arteries from insulin-resistant rats (19). There is evidence that leptin activates BKCa in rat hippocampal neurons (61). In HEK-293 cells leptin increased BKCa activity both in cells expressing the α-subunit alone and in the presence of the β1-subunit (61). Periadventitial adipose tissue has been shown to secrete an unknown factor called ADRF that is believed to act via KV, KATP, and BKCa (26). Caveolins play an important role in cell signaling and distribution of receptors, channels, and effectors (56). Interaction between caveolin-1 and BKCa affects the function and surface expression of BKCa channels (1, 9). In coronary arterioles from diet-induced obese rats, reduced protein expression of caveolin-1 was associated with enhanced BKCa activity (21). Substantial further investigation is required to determine which of these factors, or factors acting in combination, are responsible for altering BKCa β1-subunit expression in obesity.
In summary, the effect of diet-induced obesity on BKCa differs in skeletal muscle and central microvasculature. Both functional and protein expression data show that the β1-subunit is impaired in skeletal muscle arterioles but enhanced in cerebral arteries, although neither alteration affected myogenic tone over the physiological pressure range in these vessels. Increased myogenic tone at low pressures observed in skeletal muscle arterioles is likely due to vessel remodeling. Data suggest that elevated BP observed in our diet-induced obese rats is not due to increased myogenic tone of the vessels studied. Nevertheless, it is clear that diet-induced obesity altered the fundamental mechanisms by which myogenic tone, vital to control of blood flow, is regulated in these two vascular beds, specifically by altering the role of BKCa. Furthermore, the mechanisms controlling overall myogenic tone can be altered and compensate for a loss or increase in BKCa expression and function. Future studies examining the effect of diet-induced obesity on BKCa-dependent pathways may determine the mechanisms involved.
This study was supported by grants from the National Health and Medical Research Council of Australia: ID 350961(to T. V. Murphy), ID 401112 (to S. L. Sandow), and ID 455243 (to S. L. Sandow and M. J. Morris).
No conflicts of interest, financial or otherwise, are declared by the author(s).
We thank Sevvandi Senadheera for technical assistance and Maria Garcia and Merck Pharmaceutical, New Jersey, for the β1-subunit antibody.
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