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J Appl Physiol 91: 2322-2333, 2001;
8750-7587/01 $5.00
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Vol. 91, Issue 5, 2322-2333, November 2001

Anorexic effect of K+ channel blockade in mesenteric arterial smooth muscle and intestinal epithelial cells

Sharon S. McDaniel*, Oleksandr Platoshyn*, Ying Yu, Michele Sweeney, Victor A. Miriel, Vera A. Golovina, Stefanie Krick, Bethany R. Lapp, Jian-Ying Wang, and Jason X.-J. Yuan

Department of Medicine, University of California School of Medicine, San Diego, California 92103; Department of Pharmacology and Therapeutics, University of Florida College of Medicine, Gainesville, Florida 32610; and Departments of Physiology and Surgery, University of Maryland School of Medicine, Baltimore, Maryland 21201


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Activity of voltage-gated K+ (Kv) channels controls membrane potential (Em). Membrane depolarization due to blockade of K+ channels in mesenteric artery smooth muscle cells (MASMC) should increase cytoplasmic free Ca2+ concentration ([Ca2+]cyt) and cause vasoconstriction, which may subsequently reduce the mesenteric blood flow and inhibit the transportation of absorbed nutrients to the liver and adipose tissue. In this study, we characterized and compared the electrophysiological properties and molecular identities of Kv channels and examined the role of Kv channel function in regulating Em in MASMC and intestinal epithelial cells (IEC). MASMC and IEC functionally expressed multiple Kv channel alpha - and beta -subunits (Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5, Kv2.1, Kv4.3, and Kv9.3, as well as Kvbeta 1.1, Kvbeta 2.1, and Kvbeta 3), but only MASMC expressed voltage-dependent Ca2+ channels. The current density and the activation and inactivation kinetics of whole cell Kv currents were similar in MASMC and IEC. Extracellular application of 4-aminopyridine (4-AP), a Kv-channel blocker, reduced whole cell Kv currents and caused Em depolarization in both MASMC and IEC. The 4-AP-induced Em depolarization increased [Ca2+]cyt in MASMC and caused mesenteric vasoconstriction. Furthermore, ingestion of 4-AP significantly reduced the weight gain in rats. These results suggest that MASMC and IEC express multiple Kv channel alpha - and beta -subunits. The function of these Kv channels plays an important role in controlling Em. The membrane depolarization-mediated increase in [Ca2+]cyt in MASMC and mesenteric vasoconstriction may inhibit transportation of absorbed nutrients via mesenteric circulation and limit weight gain.

voltage-gated potassium channel; membrane potential; sodium-dependent glucose symport


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

OBESITY AND BEING OVERWEIGHT are associated with a high risk of morbidity from stroke, ischemic heart disease, and diabetes mellitus (1, 30). Storage of fat is controlled by a balance between energy intake and expenditure, and a very small change in this balance can result in significant weight gain (1, 30). Energy intake depends in part on the amount of digested nutrients (e.g., sugar, protein, and fat) that are absorbed by the small intestine and the amount of absorbed nutrients (e.g., glucose, amino acid, and fatty acid) that are transported to the liver and adipose tissue via mesenteric circulation. Because the triacylglycerols stored in adipose tissue are primarily synthesized from glucose and fatty acid, excessive intake of these nutrients is the major source for accumulated fat (30). Thus pharmacological intervention designed to minimize the intestinal absorption and circulatory transportation of ingested glucose, amino acids, and fatty acids would be an effective strategy for limiting body fat storage.

After a meal, intestinal mucosa exposure to glucose, protein, and fat increases intestinal blood flow to facilitate transportation of absorbed glucose and amino acid to liver and adipose tissue (10, 11). The mesenteric blood flow is controlled by the mesenteric vascular resistance, which is increased by vasoconstriction and decreased by vasodilation. An increase in cytoplasmic free Ca2+ concentration ([Ca2+]cyt), resulting from Ca2+ influx through Ca2+ channels in the plasma membrane, is a major trigger for vascular smooth muscle contraction and thus vasoconstriction (26, 35, 38). In mesenteric arterial smooth muscle cells (MASMC), membrane potential (Em) regulates mesenteric vascular contractility by governing activity of voltage-dependent Ca2+ channels (VDCC). Because Em is primarily determined by K+ permeability through plasmalemmal K+ channels, inhibition of K+ channels would induce membrane depolarization and cause mesenteric vasoconstriction by raising [Ca2+]cyt in MASMC, resulting in increased mesenteric vascular resistance to the blood flow. This ultimately may limit the transportation of absorbed nutrients to other tissues for storage.

Furthermore, the Na+-dependent uptake of glucose (42), amino acids (36), and long-chain free fatty acids (4, 5, 37) from the lumen of the intestine into intestinal epithelial cells (IEC) is driven by the transmembrane Na+ concentration gradient and the Em. Therefore, membrane depolarization (when Em becomes less negative inside of the plasma membrane) due to dysfunctional K+ channels in IEC may also inhibit the Na+-driven absorption of glucose, amino acid, and long-chain fatty acid in the intestine by reducing the transmembrane driving force for Na+.

K+ channels are ubiquitously expressed in almost all excitable or nonexcitable cells. There are several types of K+ channels described in vascular smooth muscle cells and epithelial cells (13, 21, 23, 27, 34, 43). Pharmacological blockade of voltage-gated K+ (Kv) channels with 4-aminopyridine (4-AP) induces membrane depolarization, increases [Ca2+]cyt, and causes vasoconstriction, suggesting that the activity of Kv channels regulates Em under resting conditions in smooth muscle cells (14, 15, 23, 33, 34, 44). It has been demonstrated that the anorexic drugs, fenfluramine and dexfenfluramine, in addition to inhibiting serotonin transporters (2), decrease Kv channel activity in vascular smooth muscle cells (19, 24, 40). These observations suggest that the activity of Kv channels in MASMC may play an important role in the regulation of energy intake by controlling nutrient transportation. In this study, we tested the hypothesis that blockade of Kv channels causes mesenteric vasoconstriction, which should lead to a reduction in nutrient transportation, and therefore ingestion of a Kv-channel blocker inhibits weight gain. To this end, we characterized the electrophysiological properties and molecular identities of Kv channels in MASMC and IEC and investigated the effect of K+ channel blockade on mesenteric contractility and the anorexic effect of oral ingestion of the K+ channel blocker 4-AP in rats.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell preparation and culture. Primary cultures of MASMC were prepared from Sprague-Dawley rats (44, 45). The third to fourth divisions of mesenteric arterial branches of the abdominal aorta were isolated and incubated for 20 min in Hanks' balanced salt solution with 1.5 mg/ml collagenase (Worthington). After the incubation, a thin layer of adventitia was carefully stripped off and endothelium was removed by gently scratching the intimal surface. The remaining smooth muscle was digested with 1.75 mg/ml collagenase and 0.5 mg/ml elastase (Sigma Chemical) for 45 min at 37°C. The cells were plated onto 25-mm coverslips in petri dishes (for electrophysiological and fluorescent experiments) or directly onto 10-cm petri dishes (for molecular biological experiments) and cultured in DMEM containing 10% fetal bovine serum (FBS) in a 37°C, 5% CO2, humidified incubator. The cellular purity of MASMC in primary cultures was confirmed by the specific monoclonal antibody raised against smooth muscle alpha -actin (32). Primary cultured cells were first stained with the membrane-permeable nucleic acid stain 4',6'-diamidino-2-phenylindole (DAPI, 5 µM; Molecular Probes) to estimate total cell numbers in the cultures. All the DAPI-stained cells also cross-reacted with the smooth muscle alpha -actin antibody, indicating that the cultures contained only smooth muscle cells.

The rat IEC-6 cell line was purchased from the American Type Culture Collection at passage 13. Stock cells were maintained in T-150 flasks in DMEM supplemented with 5% FBS, 10 µg/ml insulin, and 50 µg/ml gentamicin sulfate (Sigma Chemical). Flasks were incubated at 37°C in a humidified atmosphere of 5% CO2 in air. Stock cells were subcultured once a week at 1:20 and plated at 3,500 cells/cm2 on coverslips (for electrophysiological and fluorescence experiments) and 10-cm petri dishes (for molecular biological experiments) in DMEM supplemented with 5% dialyzed FBS. Passages 15-17 were used in the experiments. There were no significant changes in biological function and characterization from passages 15-17.

Electrophysiological measurements. Whole cell K+ currents were recorded with an Axopatch-1D amplifier and a DigiData 1200 interface (Axon Instruments) using patch-clamp techniques (18, 44, 45). Patch pipettes (2-4 MOmega ) were made on a Sutter electrode puller using borosilicate glass tubes and were fire polished on a Narishige microforge. Step-pulse protocols and data acquisition were performed by using pCLAMP software. Currents were filtered at 1-2 kHz (-3 dB) and digitized at 2-4 kHz with the use of the amplifier. All experiments were performed at room temperature (22-24°C). Em in single MASMC or IEC was measured in current-clamp mode (I = 0) using the patch-clamp technique.

For recording K+ currents, a coverslip containing the cells was positioned in the recording chamber (~0.75 ml) and superfused (2-3 ml/min) with the standard extracellular (bath) physiological salt solution (PSS). The PSS contained (in mM) 141 NaCl, 4.7 KCl, 1.8 CaCl2, 1.2 MgCl2, 10 HEPES, and 10 glucose, buffered to pH 7.4 with 5 M NaOH or 2 M Tris. In Ca2+-free PSS, CaCl2 was replaced by equimolar MgCl2, and 0.1 mM EGTA was added to chelate residual Ca2+. The internal (pipette) solution for recording whole cell K+ currents contained (in mM) 125 KCl, 4 MgCl2, 10 HEPES, 10 EGTA, 5 Na2ATP, buffered to pH 7.2 with 2 M Tris. 4-AP (Sigma Chemical) was directly dissolved into PSS on the day of use; the pH value of the solution was readjusted to 7.4.

Measurement of [Ca2+]cyt. In single MASMC or IEC, [Ca2+]cyt was measured by using the Ca2+-sensitive fluorescent indicator fura 2 and a digital imaging fluorescent microscopy system (16, 17). Cells were loaded with the acetoxymethyl ester form of fura 2, fura 2-AM (3 µM for 30 min) (Molecular Probes), in the dark at room temperature (22-24°C) under an atmosphere of 5% CO2-95% air. The fura 2-loaded cells on coverslips were then transferred to a recording cell chamber mounted on the microscope stage and superfused with PSS for 20-30 min to remove the extracellular fura 2, which may have diffused out of the cell after initial loading, and to allow cytosolic esterases to cleave fura 2-AM into active fura 2. Subsequent experiments were carried out at 32°C. Fura 2 fluorescence (510-nm light emission excited by 360- and 380-nm illuminations) from the cell, as well as background fluorescence, was imaged using a GEN III charge-coupled device camera (Stanford Photonics) coupled to a Carl Zeiss microscope. Fluorescent images were obtained by using a microchannel plate image intensifier (Amperex XX1381) coupled by fiber optics to the charge-coupled device camera. Image acquisition and analysis were performed with a MetaMorph Imaging System (Universal Imaging). Video frames containing images of fura 2 fluorescence from cells, as well as the corresponding background images (fluorescence from fields devoid of cells), were digitized at a resolution of 512 horizontal × 480 vertical pixels and at eight-bit gray scale by using a Matrix LC imaging board operating in an IBM-compatible personal computer. To improve the signal-to-noise ratio, four to eight consecutive video frames were usually averaged at a video frame rate of 30 frames/s. Images were acquired at a rate of one averaged image every 3 s when [Ca2+]cyt was changing, and every 60 s when [Ca2+]cyt was stable. [Ca2+]cyt was calculated from fura 2 fluorescent emission excited at 380 and 360 nm by using the ratio method (13). In most experiments, multiple (6-10) cells were imaged in single field, and one arbitrarily chosen peripheral cytosolic area (4-6 × 4-6 pixels) from each cell was spatially averaged (16).

Measurement of diameter in isolated mesenteric artery. Isolated mesenteric arteries were dissected by methods similar to those described previously (25). The mesenteric arcade was dissected from the abdominal cavity, cleaned free of blood, and placed in a temperature-controlled dissection chamber containing a dissection solution of the following composition (in mM): 145 NaCl, 5 KCl, 2.5 CaCl2, 1 MgSO4, 1 KH2PO4, 3 MOPS, 5 glucose, and 1.0% albumin (pH 7.4). The dissected arteries were transferred to a perfusion chamber that was filled with cold Krebs buffer solution. The perfusion chamber housed two glass pipettes filled with Krebs buffer. The artery was cannulated at both ends by the glass pipettes (tip diameter, 60-80 µm) and secured by 10-0 surgical suture. One pipette was attached to a pressure-regulating device (Living Systems) while the other was attached to a closed stopcock. This configuration allowed for the study of pressure-induced responses in the absence of intraluminal flow. The chamber was placed on the stage of an inverted microscope and continuously superfused with Krebs solution (in mM): 112 NaCl, 25.7 NaHCO3, 4.9 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KHPO4, 11.5 glucose, and 10 HEPES (pH 7.4; PO2 of ~100-120 mmHg at 37°C). Arterial diameters were measured using an electronic video caliper (AM Instrument, College Station, TX) connected to a closed-circuit television system. The arteries were allowed to equilibrate to initial experimental conditions (70 mmHg at 37°C) for 1 h during which time spontaneous myogenic tone developed. Arteries that showed signs of leaks or branches were not included in the study. 4-AP was diluted to a final concentration of 2 mM in the superfusate reservoir. Phentolamine (1 µM) was included in the superfusate during the entire experiment to prevent the contribution of alpha -adrenergic receptor activation by the perivascular nerves. Experiments were concluded by obtaining maximal diameters of each artery by superfusion with a buffer in which extracellular Ca2+ was replaced by 2 mM EGTA.

Measurement of isometric tension in isolated mesenteric artery. Two stainless steel hooks (10-µm diameter) were inserted through the lumen of the mesenteric arterial rings. One hook was mounted into a perfusion chamber (0.75-ml volume), and the other hook was connected to an isometric force transducer (Harvard Apparatus). Isometric tension was continuously monitored and recorded on an IBM-compatible personal computer using DATAQ data acquisition software. Resting passive tension was maintained throughout experiments at 650-700 mg, which offered the maximal tension when the rings were exposed to 50 mM K+. The rings were equilibrated for ~60 min at resting tension and then challenged two to three times with 50 mM K+-containing perfusate to obtain a stable contractile response (46). Isolated mesenteric artery rings were superfused with modified Krebs solution (at 37°C), which contained (in mM) 138 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 NaH2PO4, 5 HEPES, 1.8 CaCl2, and 10 glucose (pH 7.4). In Ca2+-free solution, CaCl2 was replaced by equimolar MgCl2, and 0.1 mM EGTA was added to chelate residual Ca2+. In high-K+ solution, NaCl was replaced by equimolar KCl to maintain osmolarity.

RT-PCR. Total RNA was prepared from MASMC and IEC by the acid guanidinium thiocyanate-phenol-chloroform extraction method and reverse-transcribed using random hexamers [pd(N)6 primer] (39). The sense and antisense primers were specifically designed from coding regions of the target channels (Table 1). The fidelity and specificity of the sense and antisense oligos were examined by use of the BLAST program. PCR was performed by a GeneAmp PCR system using AmpliTaq DNA polymerase and accompanying buffers. The first-strand cDNA reaction mixture (3 µl) was used in a 50-µl PCR reaction consisting of 0.2 nmol of each primer, 10 mM Tris · HCl, pH 8.3, 50 mM KCl, 2 mM MgCl2, 200 µM each 2-deoxynucleotide 5'-triphosphate, and 2 units Taq DNA polymerase. The cDNA samples were amplified in a DNA thermal cycler under the following conditions: the mixture was annealed at 52-61°C (1 min), extended at 72°C (2 min), and denatured at 94°C (1 min) for 20-30 cycles. This was followed by a final extension at 72°C (10 min) to ensure complete product extension. The PCR products were electrophoresed through a 2% agarose gel, and amplified cDNA bands were visualized by ethidium bromide staining.

                              
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  		Table 1.   Oligonucleotide sequences of the primers used for RT-PCR

Determination of body weight. Sprague-Dawley rats (125-150 g) were housed in an environmentally controlled room with a 12:12-h light-dark cycle and given ad libitum access to water and Teklad rodent diet (Harlan) containing a minimum of 24% crude protein, 4% crude fat, and a maximum of 4.5% crude fiber. Body weights of rats were measured by using a scale in the morning. For the fenfluramine experiment, rats were divided into two groups: the control group (n = 22) in which saline was administered intragastrically daily for 14 days and the fenfluramine-treatment group (n = 22) in which fenfluramine (25 mg/kg, Sigma Chemical) was administered intragastrically once a day for 14 days via a curved gavage needle. For the 4-AP experiment, rats were divided into two groups: the control group (n = 23) was given drinking water with no drug added, and the 4-AP group (n = 24) was given drinking water including 4-AP (2 mM, Sigma Chemical). Drugs were dissolved in drinking water on the day of use; pH values of the solutions were measured and readjusted to the pH of control water. Water bottles for both groups were changed every 2 days for 14 days.

Statistical analysis. The composite data are expressed as means ± SE. Statistical analyses were performed by using unpaired and paired Student's t-test or ANOVA and post hoc tests (Student-Newman-Keuls) when appropriate. Differences were considered to be significant when P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Electrophysiological properties of Kv channels in MASMC and IEC. K+ efflux through Kv channels greatly contributes to the regulation of Em under resting conditions in smooth muscle cells (15, 26, 29, 34, 44). Whereas [Ca2+]cyt is increased or cellular metabolism (or intracellular ATP content) is decreased, Ca2+-activated and ATP-sensitive K+ channels participate in regulating Em (27, 34). In the following experiment, Ca2+-activated and ATP-sensitive K+ channel currents were minimized by removal of extracellular and intracellular Ca2+ (plus 1-10 mM EGTA) and inclusion of 5 mM ATP in the intracellular (pipette) solution (27) to focus on the electrophysiological and pharmacological properties of Kv channels in MASMC and IEC.

Whole cell Kv currents [IK(v)] in MASMC and IEC were elicited by depolarizing the cells from a holding potential of -70 mV to a series of test potentials ranging from -40 to +80 mV (Fig. 1, A and B). The voltage threshold for IK(v) activation was between -50 and -40 mV; the currents at -40 mV were 25.8 ± 2.6 pA in MASMC and 19.1 ± 1.6 pA in IEC (P < 0.05) (Fig. 1, B and C). The averaged slope conductances of the whole cell currents elicited by test potentials between -40 and 0 mV were ~5.7 ± 1.0 nS in MASMC and 2.7 ± 0.6 nS in IEC (P < 0.05). The amplitudes of the whole cell currents in MASMC were higher than in IEC; the currents at +80 mV were 1,408.9 ± 85.6 pA in MASMC (n = 68) and 538.7 ± 31.4 pA in IEC (n = 51, P < 0.001). However, because the cell size and capacitance are quite different between MASMC and IEC (the averaged capacitance was 19.2 ± 0.8 pF in MASMC and 8.0 ± 0.3 pF in IEC) (Fig. 1D), the current density of IK(v) at +80 mV was actually indistinguishable between MASMC (73.4 ± 6.7 pA/pF) and IEC (66.6 ± 6.1 pA/pF) (Fig. 1E).


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  		Fig. 1.   Characterization of whole cell voltage-gated K+ (Kv) currents [IK(v)] in mesenteric artery smooth muscle cells (MASMC) and intestinal epithelial cells (IEC). A: representative records of superimposed currents, elicited by depolarizing the cells to test potentials ranging from -40 to +80 mV in 20-mV increments (holding potential, -70 mV). Leakage and capacitance currents were subtracted. B: current (I)-voltage (V) relationships (I-V curves, means ± SE) of the currents recorded from MASMC (, n = 68) and IEC (open circle , n = 51). C: summarized data (means ± SE) showing current amplitudes at -40 mV in MASMC and IEC. D and E: averaged cell capacitance (Cm, D) and current density of IK(v) at +80 mV (E) in MASMC (n = 42, solid bars) and IEC (n = 54, open bars). Data are means ± SE. ***P < 0.001 vs. MASMC.

The normalized whole cell currents at +80 mV, which were averaged from all MASMC and IEC tested, showed that the currents were rapidly activated and slowly inactivated (Fig. 2). The time constants for current activation were 1.2 ± 0.1 ms in MASMC and 1.4 ± 0.2 ms in IEC, whereas the time constants for current inactivation were 194.6 ± 29.3 ms and 167.1 ± 30.6 ms (P = 0.48), respectively (Fig. 2C). As shown in Fig. 3A, the IK(v) elicited by a 20-s test pulse of +60 mV (from a holding potential of -70 mV) in MASMC and IEC were composed of at least three components: a rapidly inactivating component, a slowly inactivating component, and a noninactivating component. The fast and slowly inactivating components of the outward IK(v) were activated at potentials between -60 and -40 mV and completely inactivated at potentials more positive than 0 mV (Fig. 3, B and C). The half-inactivation potentials were -30 and -40 mV, whereas the half-activation potentials were -12.5 and -15 mV, respectively, in MASMC and IEC (Fig. 3C). The window currents appeared to be at a range between -50 and -10 mV and peaked at -21 mV in MASMC and -27 mV in IEC, suggesting that all the components of IK(v) participate in the regulation of resting Em in these cells. These results indicate that the electrophysiological properties (e.g., current density, voltage threshold for current activation, and kinetics of current inactivation) of IK(v) are comparable between MASMC and IEC, suggesting that both cell types functionally express similar Kv channels.


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  		Fig. 2.   Comparison of kinetics of IK(v) in MASMC and IEC. A: averaged currents (elicited by a 300-ms test pulse of +80 mV) obtained from MASMC (left) and IEC (right) tested; the currents were normalized to the maximal amplitude of each current record and are expressed as percentage of the maximal currents (I/Imax). B: time courses of activation (left) and inactivation (right) of the averaged currents shown in A. C: time constants (means ± SE) for activation (tau act; left) and inactivation (tau inact; right) of the currents elicited by a test potential of +80 mV in MASMC (n = 34) and IEC (n = 32).



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  		Fig. 3.   Comparison of steady-state activation and inactivation kinetics of IK(V) in MASMC and IEC. A: averaged currents (elicited by a 20-s test pulse of +60 mV) obtained from 12 MASMC (left) and 16 IEC (right) tested; currents were normalized to the maximal amplitude of each current record and are expressed as percentage of I/Imax. B: representative current records showing steady-state current inactivation in MASMC (left) and IEC (right). A double-pulse protocol was used: cells were first hyperpolarized to -110 mV or depolarized to -20 mV from a holding potential of -70 mV for 2 s and then depolarized from -70 mV to +60 mV for 300 ms. C: normalized current amplitudes (I/Imax, ) plotted against conditioning potentials ranging from -110 to +60 mV (test potential was +80 mV) and normalized conductance (g/gmax, open circle ) plotted against test potentials ranging from -80 to +80 mV (holding potential was -70 mV). Data are means ± SE from 34 MASMC (left) and 43 IEC (right). Smooth lines represent best fit to inactivation and activation curves.

Molecular identification of IK(v) in MASMC and IEC. At the molecular level, Kv channels are homomeric or heteromeric tetramers composed of the pore-forming alpha  subunits and the cytosolic regulatory beta -subunits (12, 20, 21). Using RT-PCR analysis, we identified 1) seven Kv channel alpha -subunits, Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5, Kv2.1, and Kv4.3; 2) an electrically silent alpha - subunit, Kv9.3; and 3) three beta -subunits, Kvbeta 1.1, Kvbeta 2.1, Kvbeta 3, in MASMC and IEC (Fig. 4). The Kv channel alpha -subunit, Kv1.6, appeared to be expressed only in IEC but not in MASMC (Fig. 4A, middle). These results indicate that both MASMC and IEC express multiple Kv channel alpha - and beta -subunits.


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  		Fig. 4.   RT-PCR analysis of Kv channel alpha - and beta -subunits in MASMC (M) and IEC (I). PCR amplified products displayed in agarose gels stained with ethidium bromide for Kv1.1 (594 bp), Kv1.2 (295 bp), Kv1.3 (515 bp), Kv1.4 (322 bp), Kv1.5 (267 bp), Kv1.6 (394 bp), Kv2.1 (528 bp), Kv4.3 (270 bp), and Kv9.3 (568 bp) (A), as well as Kvbeta 1.1 (150 bp), Kvbeta 2.1 (150 bp), Kvbeta 3 (395 bp) (B), and beta -actin (244 bp) transcripts. M, molecular weight standard (100-bp DNA ladder).

Effects of 4-AP on whole cell IK(v) and Em in MASMC and IEC. At doses of <= 5 mM, 4-AP is a relatively specific blocker of Kv channels (3, 23, 27, 44). Indeed, extracellular application of 5 mM 4-AP significantly and reversibly reduced whole cell IK(v) in MASMC and IEC (Fig. 5). The 4-AP-sensitive components of whole cell IK(v), revealed by subtracting the currents recorded during application of 4-AP from the currents recorded under control conditions, were activated at potentials more negative than -40 mV with slope conductances of 3.1 ± 0.5 nS in MASMC (n = 8) and 1.7 ± 0.4 nS in IEC (n = 9, P < 0.001) between -40 and 0 mV (Fig. 5, A and B, insets).


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  		Fig. 5.   4-Aminopyridine (4-AP) reversibly reduces whole cell IK(v) in MASMC and IEC. A and B: representative current records, elicited by depolarizing MASMC (A) or IEC (B) to a series of test potentials ranging from -40 mV to +80 mV in 20-mV increments (holding potential, -70 mV), before (control), during (4-AP), and after (recovery) application of 4-AP (5 mM). Bottom panels show the 4-AP-sensitive currents, obtained by subtracting the currents recorded during application of 4-AP from the currents recorded under control conditions, and their current-voltage relationships (I-V curves, insets) in MASMC (A) and IEC (B). C: summarized data (means ± SE) showing amplitudes of currents (IK) at +80 mV before (open bars), during (closed bars), and after (gray bars) application of 4-AP in MASMC (n = 11, left) and IEC (n = 12, right). ***P < 0.001, **P < 0.01 vs. control and recovery.

Under resting conditions, the membrane input resistance (Rm) was very high in MASMC (9.5 ± 4.4 GOmega ) and IEC (8.1 ± 3.6 GOmega ; P = 0.84). Therefore, a small change in IK(v) would cause a large change in Em in both MASMC and IEC. Resting Em in MASMC (-45 ± 2 mV, n = 24) and IEC (-39 ± 2 mV, n = 34) were slightly but not significantly different (P = 0.36) under control conditions. Consistent with the inhibitory effect on whole cell IK(v), extracellular application of 5 mM 4-AP reversibly depolarized MASMC by ~16 mV and IEC by ~18 mV (Fig. 6A). Similar to the effect of 4-AP, increasing extracellular K+ concentration from 4.7 to 50 mM (which reduces K+ efflux and shifts the K+ equilibrium potential from -83 to -25 mV) also depolarized Em in MASMC and IEC (Fig. 6B). These results indicate that activity of Kv channels greatly contributes to the regulation of resting Em in both MASMC and IEC and that pharmacological blockade of Kv channels with 4-AP induces membrane depolarization.


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  		Fig. 6.   4-AP and 50 mM K+ reversibly depolarize MASMC and IEC. A: representative records (a) and summarized data (b) of membrane potential (Em), measured by the current-clamp (I = 0) technique, in MASMC (left) and IEC (right) before, during, and after application of 5 mM 4-AP. Data in b are means ± SE from 24 MASMC and 26 IEC. B: representative records (a) and summarized data (b) of Em in MASMC and IEC before, during, and after application of 50 mM K+ (50 K). Data in b are means ± SE from 17 MASMC and 16 IEC. ***P < 0.001, **P < 0.01 vs. control and recovery.

Effects of 4-AP-induced Em depolarization on [Ca2+]cyt in MASMC and on vasomotor tone in isolated mesenteric arteries. Because of the voltage dependence of VDCC and Na+/Ca2+ exchanger in the plasma membrane, Em serves as a major regulator of [Ca2+]cyt in smooth muscle cells (8, 9, 14, 26, 31, 33). Using RT-PCR, we detected high mRNA levels of VDCC alpha 1- and beta 1-subunits in MASMC (Fig. 7). In contrast, neither the alpha 1- nor the beta 1-subunit of VDCC was detected in IEC (Fig. 7), even when the cDNA samples were amplified for more than 35 cycles. These results suggest that VDCC are highly expressed in MASMC but not expressed in IEC.


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  		Fig. 7.   RT-PCR analysis of voltage-gated Ca2+ channel (VDCC) alpha - and beta -subunits in MASMC (M) and IEC (I). PCR amplified products displayed in agarose gels stained with ethidium bromide, for VDCCalpha 1 (347 bp), VDCCbeta 1 (549 bp), and beta -actin (244 bp) transcripts. M, molecular weight standard (100 bp DNA ladder).

In smooth muscle cells, the voltage window for sustained elevation of [Ca2+]cyt through VDCC ranges from -40 to -25 mV (14, 26, 33). Thus the 4-AP-induced Em depolarization would open VDCC and increase [Ca2+]cyt. Indeed, blockade of Kv channels with 4-AP induced membrane depolarization (Fig. 6A) and reversibly increased [Ca2+]cyt (Fig. 8, A and B) in MASMC.


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  		Fig. 8.   Blockade of Kv channels with 4-AP increases cytoplasmic free Ca2+ concentration ([Ca2+]cyt) and induces mesenteric vasoconstriction. A: representative record of [Ca2+]cyt in a MASMC (left) and summarized data (means ± SE, right) showing [Ca2+]cyt measured in peripheral areas of MASMC (n = 64), before (basal), during (4-AP), and after (wash) application of 5 mM 4-AP. ***P < 0.001 vs. basal. B: pseudocolor Ca2+ images (top panels) showing [Ca2+]cyt in the cells before, during, and after extracellular application of 4-AP, and a fura 2 fluorescence (F360) image (bottom panel) showing the cells from which [Ca2+]cyt was measured. C: schematic diagram showing the setup used to measure vessel diameter. The vessel (a) was cannulated by glass pipettes (b) and was superfused with Krebs solution in a recording chamber (c) on the microscope. The 2 vertical white broken lines in the transmitted light image (d) of a pressurized rat mesenteric artery are the video calipers used to measure lumen diameter. D: representative record (left) of the lumen diameter of an isolated rat mesenteric artery before and during application of 4-AP (2 mM). Summarized data (means ± SE, right) showing the averaged diameters of mesenteric arteries (n = 8) before (cont) and during (4-AP) 3-min treatment with 4-AP. **P < 0.01 vs. control.

In vascular smooth muscle cells, a rise in [Ca2+]cyt is a major trigger for vasoconstriction (35). Indeed, the 4-AP-induced membrane depolarization and increase in [Ca2+]cyt caused mesenteric vasoconstriction. In isolated mesenteric arterial segments, extraluminal application of 2 mM 4-AP reduced the diameter of the pressurized artery by ~20% (Fig. 8, C and D). Furthermore, 10 mM tetraethylammonium, a K+ channel blocker, also caused significant vasoconstriction; the diameter of the vessels was reduced from 125 ± 3 to 99 ± 6 µm (n = 6, P < 0.01). Because vascular resistance is inversely proportional to the fourth power of the arterial lumen radius, the 4-AP-induced mesenteric vasoconstriction would be expected to significantly increase mesenteric vascular resistance to the blood flow.

Effects of 50 mM K+ on [Ca2+]cyt in MASMC and IEC. Raising extracellular K+ concentration to 50 mM depolarized Em in both MASMC and IEC (Fig. 6B). In MASMC that highly express VDCC, the 50 mM K+-induced membrane depolarization significantly increased [Ca2+]cyt (Fig. 9A). Removal of extracellular Ca2+ abolished the high K+-mediated increase in [Ca2+]cyt (data not shown), suggesting that the [Ca2+]cyt is increased mainly by Ca2+ influx through opened VDCC in MASMC. Accordingly, extracellular application of 50 mM K+-containing solution increased isometric tension in isolated mesenteric arterial rings, whereas removal of extracellular Ca2+ reversibly blocked the high K+-induced vasoconstriction (Fig. 9B).


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  		Fig. 9.   Effects of 50 mM K+ on [Ca2+]cyt in MASMC (left) and IEC (right) and on isometric tension in isolated mesenteric arterial rings. A: representative records (a) showing time courses of the 50 mM K+-induced [Ca2+]cyt changes in MASMC and IEC. Summarized data (b, means ± SE) showing [Ca2+]cyt levels before (basal), during (50 K), and after (wash) extracellular application of 50 mM K+ in MASMC (gray bars, n = 45) and IEC (solid bars, n = 35). ***P < 0.001 vs. basal and wash. B: representative record showing time course of the 50 mM K+-induced contraction in an isolated mesenteric arterial ring (a). Ca2+-free solution (0 Ca) containing 50 mM K+ was applied to the vessel when 50 mM K+-mediated contraction reached plateau. Summarized data (b, means ± SE, n = 16) showing vessel tension before (basal), during (50 K), and after (wash) application of 50 mM K+ in the presence (1.8Ca) or presence (0 Ca) of extracellular Ca2+ (1.8 mM). ***P < 0.001 vs. solid bars.

Because VDCC are not expressed in IEC, the membrane depolarization (Fig. 6B) induced by raising extracellular K+ concentration to 50 mM negligibly affected [Ca2+]cyt (Fig. 9A). Actually, the membrane depolarization due to inhibited Kv channels attenuated Ca2+ influx through voltage-independent Ca2+ channels (e.g., store-operated Ca2+ channels) (28) in IEC by reducing the transmembrane driving force for Ca2+ (41).

Anorexic effect of blocking Kv channels by 4-AP on weight gain in rats. Because the mesenteric blood flow is required to transport absorbed nutrients from intestine to liver and adipose tissues, an increase in the mesenteric vascular resistance would lead to a decrease in nutrient transportation (10, 11) and may thus inhibit weight gain.

Body weight of the rats in the control group (n = 22-26), fed with water and normal food, rose by 55-85 g after 2 wk. Fenfluramine (25 mg/kg body wt), an appetite suppressant that has been demonstrated to block K+ channels in vascular smooth muscle cells (24), reduced weight gain in rats by 64% compared with the rats intragastrically administered with saline (Fig. 10A). Similarly, the Kv-channel blocker 4-AP (2 mM, dissolved in the drinking water) inhibited weight gain in rats by 60% compared with the rats administered only drinking water (87.3 ± 7.8 vs. 35.1 ± 7.5 g, P < 0.001) (Fig. 10B).


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  		Fig. 10.   Inhibition of Kv channels with 4-AP reduces body weight gain in rats. A: summarized data (means ± SE) showing weight gain in rats intragastrically fed with saline (control, open circle , n = 22) or fenfluramine (Fen, 25 mg/kg, , n = 22). ***P < 0.001 vs. control (Student-Newman-Keuls). B: summarized data (means ± SE) showing weight gain in rats that drank water (control, open circle , n = 23) or water containing 4-AP (2 mM, , n = 24). ***P < 0.001 vs. control (Student-Newman-Keuls).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Body weight or fat accumulation in adipose tissues is controlled by a balance of energy (food) intake and expenditure. A major function of mesenteric circulation is to transport absorbed products of digestion (e.g., glucose, amino acids, and fatty acid) to liver and adipose tissues for synthesizing triacylglycerols, an important form of accumulated fat. The transportation of the absorbed nutrients is directly proportional to the mesenteric blood flow, which mainly depends on the contractile state of mesenteric arteries. Food intake or exposure of intestinal epithelium to glucose increases mesenteric blood flow due to nitric oxide-mediated mesenteric vasodilation (10, 11), suggesting a significant contribution of enhanced mesenteric vasodilation to the transportation of absorbed nutrients to other tissues. Therefore, a reduction in mesenteric blood flow would diminish the transportation of absorbed nutrients to liver and adipose tissues and may inhibit weight gain.

In this study, we observed that MASMC and IEC similarly expressed multiple Kv channels. The whole cell IK(v) shared similar electrophysiological (e.g., current density, voltage threshold, kinetics of current activation and inactivation) and pharmacological (e.g., in response to 4-AP) properties in these two cell types. Inhibition of Kv channels by 4-AP or decrease of K+ efflux by raising extracellular K+ concentration to 50 mM induced membrane depolarization in both MASMC and IEC. Because MASMC highly express VDCC, the 4-AP- or 50 mM K+-induced membrane depolarization reversibly increased [Ca2+]cyt, caused mesenteric vasoconstriction, and reduced the diameter of mesenteric artery. The resultant decrease in the blood flow through mesenteric arterioles and capillaries would ultimately inhibit the transportation of absorbed nutrients to other tissues. In IEC, the 50 mM K+-mediated Em depolarization had little effect on [Ca2+]cyt because VDCC are not expressed in these cells. However, the membrane depolarization would reduce the transmembrane Na+ driving force required for the Na+-dependent uptake of glucose, amino acid, and long-chain free fatty acid in epithelial cells (4, 5, 36, 37, 42) and thus may contribute to inhibit absorption of the nutrients in small intestine. Indeed, 4-AP, similar to the appetite suppressant fenfluramine, significantly reduced weight gain in rats.

Role of K+ channel activity in the regulation of Em in MASMC and IEC. In both excitable and nonexcitable cells, the transmembrane K+ permeability through K+ channels is a key determinant of Em when the K+ gradient remains constant (27). It has been demonstrated that K+ currents through Kv channels are, in part, responsible for controlling Em under resting conditions in smooth muscle cells (11, 19, 24, 33). Blockade of Kv channels (e.g., by 4-AP) induces Em depolarization because of decreased IK(v), whereas opening of Kv channels (e.g., by nitric oxide) induces Em hyperpolarization because of increased IK(v) (22, 47). Both MASMC and IEC have very large membrane input resistance, and thus a very modest change in whole cell IK(V) would cause a large change in Em.

In MASMC and IEC, we identified 1) seven functional Kv channel alpha -subunits (Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5, Kv2.1, and Kv4.3), 2) an electrically silent Kv channel modulatory alpha -subunit (Kv9.3), and 3) three Kv channel beta -subunits (Kvbeta 1.2, Kvbeta 2.1, and Kvbeta 3). Additionally, Xu et al. (43) recently demonstrated that MASMC also express Kv2.2, Kv3.2, Kv3.3, Kv3.4, Kv4.1, and Kv4.2. The Kv channel alpha -subunit, Kv1.6, was the only subunit tested in our study that was highly expressed in IEC but not in MASMC (also see Ref. 43). These observations suggest that whole cell IK(V) are controlled by homomeric and heteromeric channels encoded by multiple Kv channel alpha - and beta - subunits. Which Kv channel subunits dominantly contribute to the regulation of Em in MASMC and IEC is still incompletely understood.

Role of Em in the regulation of mesenteric vascular resistance. A rise in [Ca2+]cyt in vascular smooth muscle cells is a major trigger for vasoconstriction (35). [Ca2+]cyt can be increased by Ca2+ influx through sarcolemmal Ca2+ channels and by Ca2+ release from intracellular stores (mainly the sarcoplasmic reticulum) (6-9, 38). By governing Ca2+ influx via VDCC, Em plays a critical role in regulating [Ca2+]cyt and vascular tone (26, 38). In smooth muscle cells, much of the increase in cytosolic Ca+ necessary for muscle contraction is due to Ca2+ entry through plasma membrane Ca2+ channels, dominantly VDCC (14, 26, 33, 35, 44). Because MASMC highly express VDCC, the 4-AP- or 50 mM K+-induced Em depolarization significantly increased [Ca2+]cyt and caused mesenteric vasoconstriction.

Mesenteric blood flow (Q) is decreased when the mesenteric artery constricts and is increased while the vessel relaxes, as shown by the equation Q = Delta P/MVR, where Delta P is the difference of mean arterial and venous pressure, and MVR is the mesenteric vascular resistance. Because MVR inversely varies as the fourth power of the radius of the artery (MVR = 8eta l/pi r4, where eta  is viscosity of blood and l and r are the length and radius of the vessels, respectively), the principal determinant of the vascular resistance to blood flow is the caliber of the vessels. Thus a very small decrease in the radius (or diameter) of the mesenteric arterial lumen, due to a modest vasoconstriction, would cause a significant increase in mesenteric vascular resistance and a marked decline in blood flow to the mesenteric capillaries. As shown in this study, blockade of Kv channels in MASMC by 4-AP reduced the diameter (or radius) of the isolated mesenteric arterial rings by ~20%, which would significantly decrease the blood flow through mesenteric arterioles and capillaries and subsequently inhibit the transportation of absorbed nutrients.

Possible role of Em in the absorption of glucose and amino acids in intestinal epithelial cells. Absorption of monosaccharides and amino acids from the intestinal lumen into the blood relies on import of the substances into IEC followed by export from the cells into the fluid surrounding the basolateral surface. Glucose and amino acids are mainly imported into IEC by Na+-dependent glucose or amino acid symporters located in the apical membrane (36, 42). These symporters are driven by the transmembrane Na+ concentration gradient and electrical potential difference (Em). The negative Em in IEC is a major driving force for the movement of Na+ into the cell under conditions that the transmembrane Na+ concentration gradient remains unchanged. Thus the membrane depolarization due to blocked Kv channels may also reduce the Na+ driving force in IEC, attenuate Na+-dependent glucose and amino acid uptake into IEC, and subsequently inhibit the absorption of ingested nutrients.

Possible mechanisms involved in the anorexic effects of fenfluramine and 4-AP. In addition to inhibiting serotonin transporters (2), the appetite suppressants fenfluramine and dexfenfluramine have been demonstrated to attenuate the mRNA and protein expression of Kv channel alpha -subunits (40) and decrease the whole cell IK(v) (19, 24). 4-AP is a potent blocker of K+ channels (23, 27, 44); it appears to be more selective to block Kv channels in vascular smooth muscle cells at doses of <= 5 mM (23, 27). The important roles of Kv channel activity in the regulation of Em in MASMC and IEC direct us to speculate that fenfluramine and 4-AP may similarly exert their anorexic effects on rats by blocking K+ channels. The membrane depolarization in MASMC opens VDCC, increases [Ca2+]cyt, causes mesenteric vasoconstriction, and inhibits transportation of absorbed nutrients via mesenteric circulation. The membrane depolarization in IEC decreases the transmembrane Na+ driving force that is required for the Na+-driven uptake of digested nutrients into intestinal epithelial cells and inhibits the absorption of nutrients (Fig. 11). Restriction of nutrient absorption and transportation would significantly contribute to reduce energy intake and weight gain. This study provides a new concept for developing specific blockers of K+ channels in IEC and MASMC as drugs to reduce weight gain.


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  		Fig. 11.   Schematic diagram showing the possible role of Kv channel function in the regulation of blood flow through mesenteric arteries (MA) and of glucose transport through intestinal epithelial cells. Absorption of digested food from the intestinal lumen into the blood and transportation of the absorbed nutrients via mesenteric circulation to liver and adipose tissues are 2 major processes in energy intake (A). Inhibition of Kv channels with 4-AP caused plasma membrane depolarization, which would inhibit Na+-dependent nutrient absorption by reducing the inwardly directed Na+ driving force in IEC (B) and attenuate nutrient transportation by causing mesenteric vasoconstriction and a decrease in mesenteric blood flow (C). Glu, glucose; (-), inhibition.


    ACKNOWLEDGEMENTS

We are grateful to Drs. M. P. Blaustein, Y. Cai, and X. Deng for advice and support.


    FOOTNOTES

* S. S. McDaniel and O. Platoshyn contributed equally to this work.

This work was supported in part by grants from the National Institutes of Health (HL-54043 and HL-64945 to J. X.-J. Yuan and DK-45314 and DK-57819 to J.-Y. Wang) and a Merit Review Grant from the Department of Veterans Affairs (to J.-Y. Wang). Dr. J. X.-J. Yuan is an Established Investigator of the American Heart Association.

Address for reprint requests and other correspondence: J. X.-J. Yuan, Dept. of Medicine, UCSD Medical Center, 200 W. Arbor Dr., San Diego, CA 92103-8382 (E-mail: xiyuan{at}ucsd.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. Section 1734 solely to indicate this fact.

Received 7 March 2001; accepted in final form 28 June 2001.


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V. Pucovsky, R. F Moss, and T. B Bolton
Non-Contractile Cells with Thin Processes Resembling Interstitial Cells of Cajal Found in the Wall of Guinea-Pig Mesenteric Arteries
J. Physiol., October 1, 2003; 552(1): 119 - 133.
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