INTRODUCTION

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INSULIN RESISTANCE (IR), with its associated hyperinsulinemia, is a common metabolic disorder in the adult population of prosperous societies (32) and has traditionally been defined as impairment in the signaling pathway linking insulin to its metabolic effects. A serious complication of IR is increased cardiovascular morbidity and mortality (12), with a higher incidence of both hypertension (21) and atherosclerosis (12). Moreover, IR is also associated with characteristic features that are themselves cardiovascular risk factors, including dyslipidemia characterized by low high-density lipoproteins, elevated triglycerides, and high low-density lipoproteins (11, 14, 27). Athough IR is a widely accepted risk factor for the development of hypertension and ischemic heart disease, the exact mechanism by which IR promotes vascular disease/dysfunction remains largely unknown. Several mechanisms have been proposed during the last decade to account for this impaired vascular function. For example, increased catecholamine outflow from the sympathoadrenal system is associated with hyperinsulinemia, whereas sympathectomy prevents high-fructose diet-induced hyperinsulinemia and hypertension (42). On the other hand, plasma norepinephrine levels were not altered in a group of hyperinsulinemic patients (28), and another study (41) demonstrated that heightened sympathetic activity did not contribute to IR-related hypertension.

There is substantial evidence suggesting that altered vascular reactivity in IR may be attributed to impaired endothelium-dependent vasodilation. Patients with IR exhibit depressed endothelium-mediated relaxation to increasing doses of methacholine (1, 40). Similar results were observed in patients with non-insulin-dependent diabetes (44) and microvascular angina (35), which are commonly associated with the IR syndrome (25). Moreover, animal studies have demonstrated impaired endothelium-dependent relaxation responses in both macro- and microvessels from the fructose-fed rat model of IR. Specifically, we (17) demonstrated that impaired relaxation to acetylcholine in small mesenteric arteries from IR rats develops before the onset of hypertension. Furthermore, it has been suggested that this impaired relaxation response might be attributed to reduced potassium conductance through calcium-activated potassium channels in microvascular smooth muscle; however, potential effects of IR on potassium channels in vascular myocytes are unknown. The objective of the present study was to directly examine alterations in the activity of the large conductance calcium- and voltage-activated potassium (BK_Ca) channel in vascular myocytes from IR animals. We examined the effects of IR on both the expression of this channel and its activity at both the macroscopic (whole cell) and microscopic (single channel) level. Our findings indicate that IR depresses the activity of BK_Ca channels in vascular myocytes from resistance arteries, which could impair vascular relaxation. This defect may contribute to the hypertension so often associated with the IR syndrome.

	METHODS

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Animals. Male Sprague-Dawley rats were obtained at 6 wk of age and randomized into one of two groups: IR or control animals. The IR group was fed a fructose-rich diet (containing 66% fructose, 22% casein, and 12% lard plus essential vitamins and minerals, Teklad Labs; Madison, WI), and control animals received standard rat chow. We (17) previously demonstrated that fructose-fed rats develop IR within 7 days, endothelial dysfunction within 14 days, and borderline hypertension within 20-28 days of diet therapy. Each group of animals was continued on their respective diets for a period of 4 wk. Glucose clamp experiments and glucose tolerance tests have been performed, establishing that this model does develop IR at the level of the skeletal muscle (4, 15). This was shown to correlate with fasting hyperinsulinemia in this model. In the present experiments, fasting hyperinsulinemia was used as a marker for the development of IR.

Measurement of blood pressure. One day before diet treatment was ended, rats were sedated with pentobarbital sodium (30 mg/kg ip) and ketamine (1 mg/kg ip). With use of an aseptic technique, an arterial cannula [polyethylene (PE)-10 tubing coupled to PE-50 tubing] was placed into the femoral artery for measurement of arterial pressure. The external portion of the cannula (PE-50) was tunneled under the skin, sutured between the scapula, and filled with heparinized saline solution. Animals were allowed to recover from this procedure for 24 h. During the first 18 h of the recovery period, all animals had free access to food and water. This period was followed by a 6-h fast. After the recovery period, the arterial cannula was aligned to a fluid-filled transducer (CPXL-23, Statham; Costa Mesa, CA), and the signal was conditioned, amplified, and digitized for measurement of conscious, unrestrained blood pressure. Animals were allowed to acclimatize to their new environment for 30 min before blood pressure was recorded. All data were fed on-line to an IBM personal computer and an analog-to-digital converter. Acquisitions of blood pressure were taken every 20 s for a period of 30 min. Software (DataQ, Windaq) stored the blood pressure for each experiment. These data were averaged for each animal to determine the mean resting blood pressure.

Biochemical measurements. After blood pressure measurements, rats (after a 6-h fasting period) were anticoagulated (500 units ip heparin) and anesthetized (50 mg/kg ip pentobarbital sodium). A midline incision was made, and the abdominal and chest cavities were opened. A 1-ml blood sample was drawn from the left ventricle for evaluation of fasting serum insulin and glucose concentrations. Plasma insulin was assayed by using a dextran-coated charcoal immunoassay (13). Glucose concentrations were measured using a Glucose Trinder kit (Sigma; St. Louis, MO).

Isolation of single mesenteric smooth muscle cells. Myocytes were isolated daily from third- or fourth-order mesenteric small arteries by a modification of a previously described procedure (7). Briefly, the endothelium was removed, and the adventitia of microvessels were dissected away under a microscope. The remaining smooth muscle-rich media layer was digested in an enzyme solution consisting of 6 mg papain, 4 mM dithiothreitol, 2 mg collagenase, and 0.02% bovine serum albumin. After 30 min of gentle shaking, muscle strips were lightly triturated, and the enzyme solution was diluted by the adding excess enzyme-free solution. The solution was then removed and centrifuged at 500 rpm for 12 min. The pellet was resuspended in fresh medium containing (in mM) 110 NaCl, 5 KCl, 2 CaCl₂, 2 MgCl₂, 10 HEPES, 10 NaHCO₃, 0.5 KH₂PO₄, 10 glucose, 0.49 EDTA, and 10 taurine. For patch-clamp experiments, several drops of cell suspension were placed in a microscope chamber containing the appropriate recording solution.

Ion channel recording. Whole cell currents were measured from metabolically intact cells using the amphotericin perforated patch technique (7). In contrast to standard whole cell techniques, perforated patch recordings provide current measurements with only minimal decay of the current or loss of soluble cytoplasmic components due to cellular dialysis. Furthermore, endogenous calcium-buffering systems are not inactivated by dialyzing the cell with calcium chelators, as are required during whole cell recordings. The identification and characterization of single ion channels were also assessed.

Perforated patch experiments. Cells were placed in a recording solution (22-25°C) of the following composition (in mM): 140 NaCl, 5 KCl, 1 CaCl₂, 2 MgCl₂, 20 HEPES, and 20 glucose (pH 7.4). Patch pipettes were fabricated from Corning 7052 glass (Garner Glass) using a P-87 Flaming-Brown pipette programmable puller. Pipettes with a resistance of 3 M or less were used to record ion channel activity. To measure whole cell potassium currents, the tips of patch pipettes were filled with a solution containing (in mM) 90 KCH₃SO₃, 40 KCl, 5 MgCl₂, and 20 HEPES to approximate normal cellular [K⁺] and [Cl] (pH. 7.2 with KOH). The remainder of the pipette was back filled with a similar solution to which 200 mg/ml amphotericin B (diluted with dimethyl sulfoxide) was added. Leak-subtracted currents were recorded with an Axopatch 200-B amplifier (Axon Instruments) and analyzed with pCLAMP 7.0 software. All drugs were diluted into fresh bath solution and perfused into the 0.5-ml recording chamber.

Single channel recordings. Single potassium channels were measured in cell-attached patches by filling the patch pipettes (2-5 M) with the standard bath solution and making a gigaohm seal on an intact cell. The solution in the recording chamber contained (in mM) 140 KCl, 10 MgCl₂, 0.1 CaCl₂, 10 HEPES, and 30 glucose (pH 7.4). This manipulation allows precise regulation of the patch membrane voltage by the patch-clamp amplifier and yields more accurate current measurements. In experiments measuring potassium channel activity in cell-free inside-out patches, the solution facing the cytoplasmic surface of the membrane had the following composition (in mM): 115 KCH₃SO₃, 26 KCl, 2 MgCl₂, 1 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, 0.47 CaCl₂ (pCa 7), 5.0 Mg-ATP, 0.1 GTP, and 20 HEPES (pH 7.2). The solution facing the external surface was the standard recording solution described above. Currents were filtered at 2 kHz, digitized at 10 kHz, and analyzed as described above. Channel activity was quantified by calculating the single channel open probability (NP_o) as described previously (43).

Expression of BK_Ca channels by immunoblotting. Small mesenteric arteries (similar size to those used in ion channel recordings) were manually dissected and placed in ice-cold homogenization buffer containing protease inhibitors and (in mM) 50 Tris · HCl (pH 7.7), 0.1 EDTA, 1 EGTA, 250 sucrose, 0.1% 2-mercaptoethanol, 10% glycerol, 1 phenylmethylsulfonyl fluoride, 1 pepstatin A, 2 leupeptin, and 0.1% aprotein. The vessels were homogenized with a glass/glass homogenizer, and the homogenate was centrifuged at 1,000 g for 10 min, followed by a second centrifugation at 14,000 g for 15 min at 4°C. The supernatant was used for Western blot analysis as described in detail (24). A site-specific antibody directed against amino acids 913-926 of the S9/S10 linker of the -subunit of the BK_Ca channel, used previously in rat mesenteric and other vessels, was used (22). The bound antibody was detected by enhanced chemiluminescence (Amersham), and the densities of the immunoreactive bands associated with anti-_913-926 were normalized to the -actin density in each lane (22).

Chemicals. All chemicals used in this study were obtained from Sigma.

Statistics. Means and SE were calculated for systolic blood pressure, weight, serum insulin levels, and serum glucose levels for each group. Statistical differences between the control group and the group that received the fructose-rich diet were calculated for each of the above parameters using a one-way ANOVA for multiple comparisons. All data are reported as means ± SE. The criteria for significance were P < 0.05. Mean values were based on the number of animals used in each experimental group (n).

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Animal weight, mean arterial pressure, fasting plasma insulin levels, and fasting glucose levels are provided in Table 1. There was no difference in mean fasting plasma glucose levels between the control and IR groups, which is consistent with our previous data (18) demonstrating that these animals were not yet diabetic at the time of experiments (28 days of fructose-fed diet). However, mean fasting plasma insulin levels and mean arterial pressure were elevated significantly in IR animals compared with controls. These findings are consistent with other investigations indicating that after 28 days of fructose-fed diet the animals exhibit characteristics of hyperinsulinemia and hypertension (17).

Ion channel activity. We (18) suggested previously that impaired relaxation of arteries from IR animals might be attributed to dysfunction of BK_Ca channels. In vascular smooth muscle cells (VSMC), the macroscopic outward current is conducted primarily by K⁺ efflux through BK_Ca channels. Therefore, we examined whole cell steady-state outward K⁺ currents in single myocytes isolated from IR and control animals. These experiments were performed in metabolically intact muscle cells (perforated patch technique) from mesenteric microvessels. Macroscopic K⁺ currents were generated by incremental 10-mV depolarizing steps (from 50 to +50 mV), and the family of outward currents is illustrated in Fig. 1, A and B. Currents were significantly lower in myocytes from IR animals compared with controls. Currents activated slowly and did not inactivate in either group of myocytes during the 200-ms depolarization. This kinetic profile suggested activity of BK_Ca channels. The complete current-voltage relationship of average K⁺ current densities (in pA/pF) for cells from IR and control animals is provided in Fig. 1C. The macroscopic outward current was decreased at all positive voltages in myocytes from IR animals compared with controls, with no shift in the threshold for activation. For example, maximum current density at the peak of the curve (+50 mV) was 20% less in mesenteric microvascular cells from IR animals than those from controls [21 ± 1.5 (n = 4) vs. 26 ± 0.8 (n = 4) pA/pF, respectively]. Membrane capacitance, an indicator of cell membrane area, was not significantly different between control and IR animals, averaging 15.2 ± 1 and 14.1 ± 0.9 pF, respectively.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Macroscopic K+ current is reduced in myocytes from insulin-resistant (IR) animals. Typical outward K+ currents were recorded from myocytes from control (A) and IR (B) animals. The currents were elicited by incremental 10-mV depolarizing steps from 50 to +50 mV. C: mean current-voltage relationships for peak macroscopic outward K+ current in myocytes from control and IR animals. Each point represents the mean ± SE. Membrane current is expressed as current density (in pA/pF). There was no difference in the mean membrane capacitance for cells from control (15.2 ± 1 pF) and IR (14.1 ± 0.9 pF) animals.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Macroscopic large conductance voltage- and Ca2+-activated K+ (BKCa) channel [tetraethylammonium (TEA) sensitive] current is reduced in myocytes from IR animals. Typical K+ current traces were recorded in myocytes from control (A) and IR (B) animals. The currents were elicited by incremental 10-mV depolarizing steps from 50 to +50 mV before and after TEA (1 mM). TEA blocked a small component of the outward current in IR myocytes, whereas a large current component was inhibited by TEA in control cells. C and D: current-voltage relationships normalized for current density showing the effects of TEA (1 mM) on peak macroscopic K+ current in control (C) and IR (D) myocytes, respectively. The component of the total macroscopic outward current attributed to opening of BKCa channels is significantly less in cells from IR animals compared with controls.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Macroscopic BKCa channel [iberiotoxin (IbTX) sensitive] current is reduced in myocytes from IR animals. Typical K+ current traces recorded in myocytes from control (A) and IR (B) animals. The currents were elicited by incremental 10-mV depolarizing steps from 50 to +50 mV before and after IbTX (100 nM). IbTX (100 nM) blocked a small component of the outward current in IR myocytes, whereas a large current component was inhibited by IbTX in control cells. C and D: current-voltage relationships normalized for current density showing the effects of IbTX (100 nM) on peak macroscopic K+ current in control (C) and IR (D) myocytes, respectively. As observed with 1 mM TEA, the component of the total macroscopic outward current attributed to opening of BKCa channels is significantly less in cells from IR animals compared with controls.

NS-1619 and K⁺ channel activity. Superfusion of control cells with NS-1619 (10 µM), a selective BK_Ca channel activator, markedly increased the macroscopic K⁺ current in myocytes from control rats by almost twofold (from 370 to 640 pA; Fig. 4). Typical current traces before and after NS-1619 in mesenteric microvascular myocytes from control and IR animals are illustrated in Fig. 4A. The maximum NS-1619-stimulated current generated at +50 mV was 700 pA in control cells; however, the response of myocytes from IR animals to the channel agonist was significantly blunted. On average, NS-1619 increased outward current in myocytes from IR rats by 21 ± 5.4% (n = 4), whereas the increase observed in controls was 63.7 ± 3.4% (n = 4). The complete current-voltage relationship for current density from control and IR cells before and after NS-1619 is illustrated in Fig. 4, C and D.

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Activation of K+ currents by NS-1619 is greater in control cells than IR cells. Typical K+ current traces were recorded in myocytes from control (A) and IR (B) animals. The currents were elicited by incremental 10-mV depolarizing steps from 50 to +50 mV before and after NS-1619 (10 µM). NS-1619 is considered a selective BKCa channel activator. NS-1619 increased the outward K+ current to a significantly greater extent in control myocytes compared with cells from IR animals. C and D: current-voltage relationships for normalized current density showing the effects of NS-1619 (10 µM) on peak macroscopic K+ current in control (C) and IR (D) myocytes, respectively. NS-1619 only minimally activated BKCa channels in myocytes from IR animals compared with controls.

BK_Ca channel -subunit expression. The decreased macroscopic current observed in IR animals could result from decreased channel expression; therefore, we examined the expression of the -subunit of the BK_Ca channel in mesenteric smooth muscle membranes from control and IR animals. Previous control studies (20, 24) have established the specificity of the primary antibody anti-_913-926 for its recognition site on the -subunit of the BK_Ca channel. A typical immunoblot of -subunit expression is illustrated in Fig. 5. Lanes were loaded with either control or IR membrane proteins and revealed no apparent difference in the density of the 125-kDa immunoreactive band. Stripping and rehybridization of the membranes with the monoclonal antibody for the 42-kDa protein -actin revealed the same signal density for this internal standard, demonstrating uniformity of lane loading. The density signal of the -actin internal standard was not different between the control and IR preparations. The data average from different Western blot experiments using membranes from different rats indicated that the density of the 125-kDa immunoreactive band (expressed as a percentage of the -actin signal) was similar in control and IR animals.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Mesenteric BKCa channel -subunit expression is not altered in IR animals. A: comparison of the expression levels of the BKCa channel -subunit between mesenteric smooth muscle membranes from control (C) and IR animals. These experiments were performed using the anti-913-926 antibody. The antibody is a sequence-directed antibody against amino acids 913-926 on the S9/s10 linker of the -subunit of the BKCa channel. A representative experiment in which the adjacent lanes were loaded with membrane protein (10 µg) from control and IR vessels is shown. Stripping and rehybridization of the membranes with the monoclonal antibody for the 42-kDa protein -actin was used as an internal standard and showed similar expression and uniformity between lanes. KCa channel, Ca2+-activated K+ channel. B: densities of the immunoreactive bands associated with anti-913-926 expressed as a percentage of the -actin density for each lane. Data averaged from 3 separate comparisons using mesenteric microvascular arterial tissues from 3 control and 3 IR animals indicated that there was no difference in the density of the 125-kDa immunoreactive band between control and IR animals.

Single channel properties. We next examined the possibility that altered single channel properties contributed to the decrease in outward current in mesenteric microvessels myocytes from IR animals. Unitary current amplitude was plotted as a function of membrane potentials (60 and +60 mV) in Fig. 6. The resulting microscopic current-voltage relationship yielded a mean single channel slope conductance of 142.6 ± 4 pS (n = 3 animals and 6 patches) and 134.3 ± 6 pS (n = 3 animals and 6 patches) for cells from control and IR animals, respectively, and these values were similar to BK_Ca channel conductances reported previously (10). The recordings of single channel currents obtained at various membrane potentials were similar for inside-out patches from control and IR rats (data not shown).

View larger version (26K): [in this window] [in a new window]   Fig. 6.   Single BKCa channel conductance in myocytes from IR animals is unaltered. A: recordings of inside-out patches (+60 to 60 mV) obtained from microvascular myocytes of either control or IR rats under conditions of symmetrical (140 mM) [K+]. Dashed line, baseline (closed) state. B: average single channel current-voltage relationship for channel activity recorded from inside-out patches as in A. A linear fit revealed unitary conductances for control and IR animals of 142.6 ± 4 and 134.3 ± 6 pS, respectively (n = 6 patches).

View larger version (19K): [in this window] [in a new window]   Fig. 7.   Voltage and calcium sensitivity of BKCa channels is unaltered in the IR state. A: comparison of the calcium sensitivity of BKCa channels in inside-out patches from control (con) and IR mesenteric myocytes. Each point represents the mean ± SE. The probability of single channel opening (NPo) is plotted as a function of calcium concentration for BKCa channel opening at +60 mV. There was no difference in the calcium sensitivity of BKCa channel opening in membrane patches from control and IR animals. B: comparison of the voltage sensitivity of BKCa channels in inside-out patches from control and IR mesenteric myocytes. NPo is plotted as a function of membrane voltage obtained at 104 M calcium. There was no difference in the voltage dependency for BKCa channel opening in patches from control and IR myocytes.

	DISCUSSION

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IR syndrome is characterized by high risk for the development of non-insulin-dependent diabetes and accompanying vascular diseases including hypertension, atherosclerosis, and vasospastic angina (37). These vascular disorders have two common features: relative resistance to insulin-mediated glucose uptake (8) and vascular endothelial dysfunction (6, 44); however, our understanding of how hyperinsulinemia affects VSMC is far from complete. Although insulin has little effect on glucose uptake into vascular cells (14), it has a variety of other actions in VSMC. Insulin potentiates the effects of several growth factors, i.e., platelet-derived growth factor and mitogen-activated protein kinase, to promote vascular smooth muscle proliferation (2), production of extracellular matrix, and cell migration (14). Functionally, insulin has also been implicated as a vasoactive hormone. A recent report (8) suggests insulin mediates vasodilation via an endothelium-dependent mechanism. On the other hand, insulin can directly decrease intracellular Ca²⁺ levels in VSMC, causing relaxation and reduced reactivity to vasoconstrictors in the absence of endothelium (16). Potential mechanisms accounting for these vascular effects include stimulation of the Na⁺/H⁺ exchanger and Na⁺-K⁺-ATPase, leading to hyperpolarization of the cell membrane and consequent closure of voltage-gated Ca²⁺ channels (8). Thus, insulin may relax vascular smooth muscle via endothelium-dependent and -independent mechanisms, leading to an interesting conundrum observed in IR patients: Why does hypertension develop in the presence of excess vasodilator (i.e., insulin)?

The specific etiology of hypertension and atherosclerosis in the IR state has not been clearly defined. One possibility is that endothelial dysfunction occurs at a very early stage in the IR syndrome (26), and the loss of endothelial function promotes vasoconstriction and vascular smooth muscle growth (34, 39). Indeed, recent clinical and animal studies support this contention. IR patients exhibit impaired endothelium-mediated vasodilation (40), as do small mesenteric arteries from IR animals (18). Because endothelium-derived relaxing factors hyperpolarize VSMC by opening smooth muscle potassium channels (7, 29), it has been suggested that the impaired endothelium-dependent relaxation in IR might be related to abnormal potassium channel activity in VSMC (18); however, the potential effects of IR on potassium channel function are unknown. With the use of a multifaceted approach (i.e., patch clamp and Western blots), we now provide direct evidence that IR impairs BK_Ca channel function in VSMC from mesenteric microvessels.

It has become increasingly apparent that VSMC potassium channels are affected by a variety of pathological states that alter the integrity and/or excitability of vascular smooth muscle. Like many smooth muscle cells, myocytes from small mesenteric arteries exhibit substantial outward K⁺ currents (7), leading to membrane repolarization and relaxation by closing voltage-dependent Ca²⁺ channels. Although several distinct K⁺ channels are expressed in VSMC (5), the predominate type expressed in mesenteric artery smooth muscle is the BK_Ca channel (7, 10). These channels are identified by their selectivity and large single channel conductance as well as their sensitivity to membrane voltage and intracellular [Ca²⁺] (9). In the present study, the macroscopic outward K⁺ current density was significantly attenuated in mesenteric myocytes from IR animals compared with controls. More specifically, the TEA- or IbTX-sensitive component of the macroscopic K⁺ current was markedly less in myocytes from IR rats. These findings suggest that the contribution of BK_Ca channels to the outward current is significantly reduced in IR and probably accounts for the reduced current density observed in myocytes from IR animals. It is also possible that the reduction in outward current might reflect alteration of other potassium channels (e.g., ATP-sensitive K⁺ or voltage-dependent K⁺ channels); however, it is clear that BK_Ca channels predominate in mesenteric microvascular myocytes and that influence of these channels is suppressed in the IR state. Furthermore, the stimulatory effect of a BK_Ca channel activator, NS-1619, was substantially reduced in myocytes from IR animals. Thus the initial findings of the present study, i.e., that IR myocytes showed a diminished BK_Ca channel current, could clarify the cellular basis for impaired vasorelaxation and development of hypertension in the IR state. These findings are supported by previous studies (36) indicating changes in cardiac K⁺ currents in hyperinsulinemic conditions but contrast with those of Liu et al. (23) reporting increased potassium current in the aorta from aldosterone-salt hypertensive rats. This apparent discrepancy probably reflects differences in the macrovascular (aorta) vs. the microvascular preparations employed in the present study. Interestingly, insulin stimulates calcium-dependent outward K⁺ conductance of bovine retinal capillary pericytes (3); although these cells are not VSMC, they exhibit electrical properties similar to smooth muscle and are thought to be involved in regulation of the retinal microcirculation. Therefore, the BK_Ca channel appears to be an important molecular target of insulin action.

Further experiments were performed to explore the mechanism responsible for the decrease in total membrane current density in mesenteric myocytes from IR animals. Changes in macroscopic current reflect the contribution of channel number or expression (N), unitary current amplitude (i), and channel open state probability (P_o). Thus N, i, and P_o are distinct parameters that could be altered in the IR state to account for the decrease in macroscopic BK_Ca current density. With the use of a site-directed antibody targeted against the S9/S10 linker of the BK_Ca channel (20), we detected no difference in the density of a 125-kDa immunoreactive band in membranes from IR animals compared with controls. The 125-kDa band corresponds to the known molecular size of the -subunit of the BK_Ca channel (19). These findings suggest that the decreased current density in myocytes from IR animals is not related to altered expression of the pore-forming subunit of the BK_Ca channel. An important caveat, however, must be considered in interpreting these data from Western blots. These experiments detect protein expression, and it is difficult to account for potential effects of IR on posttranslational modification or trafficking of BK_Ca channel proteins between the cytoplasm and the plasma membrane. Therefore, while these immunoblot studies certainly suggest no changes in overall BK_Ca channel expression in IR animals, they cannot rule out potential effects of IR that could render channel complexes nonfunctional. In contrast, previous studies (24) have reported that VSMCs from hypertensive animals (spontaneously hypertensive rats) exhibit a significant increase in the density of a 125-kDa immunoreactive band compared with normotensive animals. In IR animals, blood pressure does not increase until around 28 days after induction of the IR state (17). Because we employed 28-day IR animals that were borderline hypertensive, we did not expect to see a similar increase in expression. Therefore, the suppression of BK_Ca current observed in myocytes from IR animals may be related to altered channel function, and subsequent patch-clamp studies were performed to address this possibility.

We did not observe a difference in single channel current amplitude in VSMC cell patches from IR or control animals, nor was there a difference in the calculated single-channel slope conductance (Fig. 5). These findings strongly suggest that decreased current density observed in myocytes from IR rats cannot be attributed to altered single channel amplitude (i). The other factor that would decrease macroscopic BK_Ca current is a lower P_o of the BK_Ca channel. In IR, the -subunit may exhibit decreased gating sensitivity or defective coupling to accessory subunits affecting voltage and/or calcium sensitivity, thereby reducing current density independent of changes in channel expression (24). For example, the -subunit of the BK_Ca channel affects the Ca²⁺ sensitivity (33). Thus if the IR state altered the expression or function of the -subunit, this should manifest as altered sensitivity of channel P_o to intracellular [Ca²⁺]. These two main determinants of the BK_Ca channel P_o, i.e., the ability to sense changes in transmembrane voltage and intracellular Ca²⁺, were examined in the single channel experiments. These studies revealed a similar relationship between voltage and single BK_Ca channel P_o in myocytes from IR or control animals. Furthermore, the sensitivity of channel P_o to intracellular [Ca²⁺] was also similar. It has been suggested that the conductance and voltage dependence of BK_Ca channels are determined by the NH₂-terminal core, whereas Ca²⁺ sensitivity appears to involve a region of several negatively charged residues at the COOH-terminal core (20). Therefore, our results suggest that the NH₂- and COOH-terminal cores of the BK_Ca channel appear to be functionally intact in myocytes from IR animals, further suggesting that IR probably does not produce significant alterations in the structure or coupling of the BK_Ca channel protein complex.

However, one must be cautious in extrapolating the results from these experiments on excised patches to intact cells or tissue. In our inside-out patch experiments, the concentration of free "intracellular" Ca²⁺ is carefully regulated; however, in intact cells, the localization and amount of activator calcium available for channel activation is unknown. It is feasible that the availability of calcium required for channel activation is reduced in the IR state, leading to decreased channel P_o and a depression of macroscopic BK_Ca current density. In support of this hypothesis, previous studies (3) have reported that insulin inhibits agonist-induced Ca²⁺ transients, possibly by closing voltage- and receptor-operated Ca²⁺ channels, inhibiting inositol (1,4,5)-trisphospate-induced Ca²⁺ release, or stimulating sarcolemmal Ca²⁺-ATPase to enhance transmembrane Ca²⁺ efflux. Furthermore, it has been shown that calcium transients or sparks may be a source of activator calcium to stimulate BK_Ca channel opening (30). It is possible that IR-induced depression of calcium release mechanisms and/or calcium spark activity could explain the present findings, and such depression of calcium release has been demonstrated in myocardial cells from streptozotocin-diabetic rats (31); however, potential effects of IR on ryanodine receptor function are unknown. Thus further experiments are required to clearly define the molecular mechanism responsible for the decreased BK_Ca current density we observed. Nonetheless, the present results suggest that the depression of potassium current in VSMC from IR animals is not related to a decrease in channel expression or a change in the biophysical properties (i.e., conductance and voltage and calcium sensitivity) of single BK_Ca channels.

In summary, our data clearly indicate that BK_Ca channel activity is reduced in mesenteric small arteries from fructose-fed IR rats. Although this is a dietary-dependent model of IR, animals exhibit all the typical clinical characteristics of IR, including hyperinsulinemia, glucose intolerance, increased triglycerides, and low high-density lipoprotein-cholesterol, as we have previously reported (26). We propose that this mechanism may contribute to impaired vascular relaxation and the development of hypertension in the IR state. Further experiments are necessary to investigate the time progression of changes in channel function in the IR state and to define the molecular nature of these alterations.