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(Circulation Research. 2000;87:496.)
© 2000 American Heart Association, Inc.

Cellular Biology

Expression and Function of Endothelial Ca²⁺-Activated K⁺ Channels in Human Mesenteric Artery

A Single-Cell Reverse Transcriptase–Polymerase Chain Reaction and Electrophysiological Study In Situ

Ralf Köhler, Christiane Degenhardt, Meike Kühn, Norbert Runkel, Martin Paul, Joachim Hoyer

From the Departments of Nephrology (R.K., C.D., M.K., J.H.), Clinical Pharmacology (M.P.), and Surgery (N.R.), Benjamin Franklin Medical Center, Freie Universität, Berlin, Germany.

	Abstract

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Abstract—Ca²⁺-activated K⁺ (K_Ca) channels have been suggested to play a role in the control of endothelial functions such as regulation of vascular tone and cell proliferation. We established a method for single-cell reverse transcriptase–polymerase chain reaction analysis in combination with the patch-clamp technique to characterize K_Ca channel expression and function in single endothelial cells (ECs) within the endothelial monolayer of intact human mesenteric arteries (MAs) and in disease states. We tested whether endothelial K_Ca channel expression and function are altered in MAs obtained from patients with colonic adenocarcinoma (CA) compared with those in MAs from non–cancer patients with inactive diverticulitis. Expression of the intermediate-conductance K_Ca channel (hIK1) was detected in non–cancer and CA patients. In whole-cell patch-clamp measurements, only ECs expressing hIK1 exhibited corresponding K_Ca currents, whereas respective K_Ca currents were missing in hIK1-negative ECs. This heterogeneity of hIK1 expression patterns is indicative of a specialized subset of ECs within the endothelial monolayer. In CA patients, compared with non–cancer patients, a 2.5-fold increase in hIK1-expressing ECs per MA was observed (P<0.05). However, K_Ca current densities in hIK1-expressing ECs of both groups were similar. In addition to hIK1, expression of the large-conductance K_Ca channel (hSlo) was detected in single ECs from CA patients. The increased K_Ca channel expression in CA patients resulted in a 2.7-fold increase of bradykinin-induced endothelial hyperpolarization compared with controls (P<0.05). This increased expression and function of K_Ca channels might indicate an altered functional state of the endothelium in cancer patients and could play a role in tumor angiogenesis.

Key Words: single-cell RT-PCR • endothelium • Ca²⁺-activated K⁺ channels • colonic cancer

	Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

 
The endothelium plays a pivotal role in a variety of vascular functions such as blood pressure control,¹ vascular remodeling,² and angiogenesis.³ In the control of endothelial function itself, ion channels play a key role by regulating endothelial membrane potential and Ca²⁺ homeostasis in response to humoral stimulation.^{4 5} Particularly, Ca²⁺-activated K⁺ (K_Ca) channels are thought to mediate endothelial hyperpolarization in response to humoral stimulation.⁶ Endothelial hyperpolarization provides the electrochemical driving force for Ca²⁺ entry, which is important for both the Ca²⁺-dependent synthesis of vasodilating factors⁷ and gene expression.⁸

In vivo, the endothelium is a highly specialized cell monolayer at the interface between blood and tissue. The specialized functions of this endothelial cell (EC) layer could be considerably altered by cell isolation and culture. Thus, the function of ECs kept under artificial cell culture conditions does not necessarily reflect the functional state of the endothelium in highly differentiated blood vessels. Particularly, the function and molecular identity of K_Ca channels have not yet been elucidated in endothelium in situ despite their importance in the control of vascular function.

We therefore performed a study to identify and characterize K_Ca channel expression in endothelium of intact human mesenteric artery (MA) preparations. For this purpose, we adapted a single-cell reverse transcriptase–polymerase chain reaction (RT-PCR) approach in combination with the patch-clamp technique. This allows a simultaneous analysis of gene expression combined with the functional study of ion-channel function in small-vessel specimens in situ by avoiding alterations due to cell isolation and culture. In the present study we demonstrate expression of the human intermediate-conductance K_Ca (hIK1) and large-conductance K_Ca (hSlo) genes and its specific pattern within the ECs of intact MAs.

Endothelial function is disturbed in disease states such as hypertension, atherosclerosis, and angiogenesis.^{3 9} Regarding endothelial function during angiogenesis, there are indications for the involvement of endothelial ion-channel function and Ca²⁺ homeostasis in the initiation of angiogenesis in response to angiogenic factors.^{10 11} We tested whether expression and function of endothelial K_Ca channels of MAs are altered in patients with colonic adenocarcinoma (CA) compared with non–cancer patients.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Harvesting of Single ECs From Human Mesenteric Arteries
Third-order MAs of 0.5 to 1 cm in length and 0.4 to 1.1 mm in outer diameter were excised out of the arterial arcades from colon specimens of patients subjected to hemicolectomy. Tissue collection was performed according to the guidelines of the local ethics committee. Vessel slices of 2 mm² were fixed on a glass capillary and placed in the experimental chamber. For cell-harvesting procedures, vessel slices were preincubated with 0.05% trypsin and 0.02% EDTA in PBS without Ca²⁺/Mg²⁺ for up to 15 minutes. A 5-minute superfusion with PBS with Ca²⁺/Mg²⁺ was performed to stop the enzymatic reaction and wash out remaining trypsin and contaminating cells. Under microscopic control, a slightly rounded EC was then selectively fixed by aspiration at the tip of the patch pipette and mechanically detached from the vessel wall.

Reverse Transcription
ECs and contents (6 µL) of the pipette were expelled into a tube containing 1 µL first-strand buffer (Life Technologies), 0.5 µL dNTPs (10 mmol/L each; Promega), 1 µL random-hexamer primer (100 µmol/L; Boehringer Mannheim), 1 µL DTT (0.1 mol/L; Life Technologies), and 0.5 µL RNase inhibitor (40 U/µL; Promega). After 1 freeze-thaw cycle to induce breakdown of the cell membrane, 0.5 µL SuperScript RT (200 U/µL; Life Technologies) was added, and the final volume (10 µL) was allowed to reverse transcribe for 1 hour at 37°C. To exclude contamination by nonendothelial mRNA, samples of bath solution were aspirated next to the ECs.

Polymerase Chain Reaction
A modified single-cell RT-PCR was performed as described previously.¹² The most efficient PCR conditions, ie, primer combination with maximal sensitivity, MgCl₂ concentration, number of cycles, and annealing temperature, were determined by using a serial dilution of human MA cDNA. First and nested primer pairs (TIB MOL BIOL) for von Willebrand factor (vWF) and endothelial NO synthase (eNOS) were selected to extend over intronic sequences. First and nested primer pairs for hIK1 and hSlo were selected that yielded no PCR product of expected size from genomic DNA of cell samples processed without reverse transcription and from 50 ng/µL human DNA after 2 PCR rounds. GenBank accession Nos. were the following: hIK1, AF022797; hSlo, U13913; vWF, K03028; and eNOS, L26914.

In a single-cell sample, cDNA of hIK1 and hSlo were coamplified along with the vWF cDNA as an EC marker and with myosin heavy chain (MyHC, X69292) cDNA to exclude contamination with smooth muscle cell cDNA. A first multiplex PCR was performed in a final volume of 50 µL containing 5 µL PCR buffer (10x), 2 µL dNTPs (10 mmol/L each), 3 µL MgCl₂ (50 mmol/L), 1 µL of each sense and antisense primer (10 pmol), 10 µL RT product, and 0.5 µL Taq DNA polymerase (5 U/µL; all Life Technologies) using a Biozym Maxicycler PTC 9600. Samples were incubated for 5 minutes at 94°C, followed by 50 cycles of 30 seconds at 94°C, 1 minute at 55°C, and 1 minute at 72°C and a final elongation for 10 minutes at 72°C. In a second multiplex PCR round with nested primers, 5 µL of the first PCR product was used for reamplification (45 cycles with annealing temperature 60°C). Amplified cDNAs were analyzed on a 2% agarose gel containing ethidium bromide. A 50-bp DNA ladder (Life Technologies) was used for molecular weight markers. Identities of the PCR products were confirmed by restriction site analysis and by sequencing using an ABI 377 automatic sequencer (ABI Prism). Primer pairs were as follows: for hIK1, first, 5'-GAGAGGCAGGCTGTTAATGC-3' (positions 1075 to 1094) and 5'-TGAGACTCCTTCCTGCGGAGT-3' (positions 1400 to 1381), and nested, 5'-CATCACATTCCTGACCATCG-3' (positions 1131 to 1150) and 5'-ACGTGCTTCTCTGCCTTGTT-3' (positions 1289 to 1270); for hSlo, first, 5'-GGACTTAGGGGATGGTGGTT-3' (positions 3237 to 3256) and 5'-GGGATGGAGTGAACAGAGGA-3' (positions 3533 to 3514), and nested, 5'-TTTACCGGCTG-AGAGATGCT-3' (positions 3314 to 3333) and 5'-GTGGGA-GGAATGGGACAG-3' (positions 3487 to 3469); for human vWF, first, 5'-GTTGTGGGAGATGTTTGCCT-3' (positions 61 to 80) and 5'-TGGAGTACATGGCTTTGCTG-3' (positions 426 to 407), and nested, 5'-CACCATTCAGCTAAGAGGAGG-3' (positions 89 to 119) and 5'-GCCCTGGCAGTAGTGGATA-3' (positions 398 to 380); for eNOS, first, 5'-ATGTTTGTCTGCGGCGATGT-3' (positions 3382 to 3401) and 5'-AGGGGCTGTTGGTGTCTGAG-3' (positions 3661 to 3642), and nested, 5'-ATGGCAACCAACGTCCTG-3' (positions 3406 to 3423) and 5'-AAAAGCTCTGGGTGCGTATG-3' (positions 3586 to 3567); and for MyHC, first, 5'-AAGTTCAAGTCCACCATCGC-3' (positions 2159 to 2178) and 5'-GAGTGTCCGTTTCCTCCTCA-3' (positions 2611 to 2590), and nested, 5'-GCCAAGATTGCACAGCTAGA-3' (positions 2189 to 2209) and 5'-GCCTGAGCTTGCTCTTGAGT-3' (positions 2516 to 2497).

Patch-Clamp Experiments
Membrane currents were recorded with a nEPC-9 (HEKA) patch-clamp amplifier and low-pass filtered (-3 dB, 1000 Hz) at a sample time of 0.5 ms.¹³ The endothelial membrane potential was recorded in the current-clamp mode. Patch pipettes had a tip resistance of 2 to 4 MOhm in symmetrical KCl solution. If not otherwise stated, the pipette solution contained (in mmol/L) KCl 135, MgCl₂ 4, EGTA 1, and HEPES 5 (pH 7.2). In some experiments the pipette solution additionally contained 1 mmol/L CaCl₂ ([Ca²⁺]_free=13 µmol/L). The NaCl bath solution contained (in mmol/L) NaCl 137, Na₂HPO₄ 4.5, KCl 3, KH₂PO₄ 1.5, MgCl₂ 0.4, and CaCl₂ 0.7 (pH 7.4). The KCl bath solution contained (in mmol/L) KCl 140, MgCl₂ 1, CaCl₂ 1, and HEPES 10 (pH 7.4). For excised inside-out patches, the cytosolic bath solution contained (in mmol/L) KCl 140, MgCl₂ 1, HEPES 10, and EGTA 10 (pH 7.2), with CaCl₂ added at concentrations yielding the calculated [Ca²⁺]_free specified. All experiments were performed at 37°C. Data analysis was performed as described previously.¹⁴ Leak currents were not subtracted before or during data acquisition. All chemicals and toxins were obtained from Sigma.

Cancer and Non–Cancer Patients
For comparison of endothelial K_Ca channel expression in disease states, MA specimens of the arterial arcades were obtained from patients undergoing colon resection for adenocarcinoma (n=8) and for stenosis (n=7) due to non–cancer reasons (diverticulitis or nonspecific mucosal inflammation) as a control group. MAs from cancer patients were prepared within an area 5 cm adjacent to the tumor. Colon specimens from cancer and non–cancer patients were histologically analyzed by the local pathologist. Colon specimens from 4 patients with diverticulitis and 1 patient with nonspecific mucosal inflammation showed no signs of pericolonic inflammation at surgery. From 2 patients with diverticulitis, which exhibited persisting signs of pericolonic inflammation at surgery, MAs were prepared distantly from the affected tissue. Characteristics of each patient from the respective groups are summarized in the Table.

View this table: [in this window] [in a new window]   Table 1. Characteristics of Cancer and Non–Cancer Patients

Statistical Analysis
The Mann-Whitney U test was used to assess differences between the 2 patient groups. Probability values of P<0.05 were considered significant. If not otherwise stated, data are given as mean±SE.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Single-Cell RT-PCR in Intact Endothelium of Human MAs
cDNA samples of 180 single cells from the luminal vessel wall of freshly dissected intact MAs were analyzed for gene expression. Detection of a specific RT-PCR product for the EC-specific vWF gene in 146 of 180 (81%) samples indicates the high efficiency of mRNA harvesting (Figure 1A). The 34 vWF-negative samples yielded no PCR products at all. These negative samples are thus most likely explained by a cell loss during transfer from the experimental bath to the RT tube rather than by the harvest of nonendothelial cells.

View larger version (58K): [in this window] [in a new window]   Figure 1. Representative RT-PCR analysis of single EC from human MA. A, Ethidium bromide–stained gels of RT-PCR products of vWF from 9 different EC probes. B, Coexpression of vWF and eNOS mRNA in 6 different EC probes detected by multiplex RT-PCR. MyHC expression was not detectable.

Because of its high sensitivity, the single-cell RT-PCR is particularly vulnerable to giving false-positive RT-PCR signals by minimal contamination from nonendothelial mRNA. Therefore, every fifth sample was a medium sample (n=48) aspirated near the endothelial surface. None of these medium samples yielded a positive PCR signal. To rule out a harvest of vascular smooth muscle cells or contamination by mRNA from leaking vascular smooth muscle cells, we tested all samples for MyHC gene expression by the multiplex RT-PCR technique. An RT-PCR signal for the MyHC cDNA was not codetected in any of the cell samples (0 of 180). An eNOS-specific PCR product was codetected in 62 of 77 (80%; in CA patients, 81%, and in non–cancer patients, 79%) vWF-positive cell samples (Figure 1B). eNOS expression was never detected in vWF-negative samples. The high rate of eNOS coexpression indicates the ability of NO formation of these ECs and thus indicates preserved endothelial function of these vessels.

K_Ca Channel Function and Expression in Single ECs
Before single-cell RT-PCR analyses, we performed single-channel and whole-cell patch-clamp experiments in electrically uncoupled single ECs to verify channel function. For identification of K_Ca currents, cells were dialyzed with a pipette solution containing 13 µmol/L [Ca²⁺]_free. In a subset of ECs, dialysis with Ca²⁺ induced a hyperpolarizing outward current. The reversal potential (V_rev) extrapolated from current-voltage relations shifted by -39±5 mV (n=7) in non–cancer patients and by -49±2 mV (n=9, P=0.08) in CA patients toward the potassium equilibrium potential, thus indicating K⁺ channel activity (Figure 2A). In a series of experiments, in which Cl^– was substituted by equal amounts of aspartate, membrane potential shifted to a similar extent by –40±4 mV (n=6) in non–cancer patients, thus indicating no considerable contribution of Ca²⁺-activated Cl^- channels to Ca²⁺-activated currents in ECs from MA. The Ca²⁺-activated cell current determined at a holding potential of 0 mV after subtraction of leak currents measured at break-in and standardized to cell capacity was 12±2 pA/pF in non–cancer patients and 16±4 pA/pF in CA patients (P=0.53) at 0 mV in 4.5 mmol/L K⁺ bath solution and 140 mmol/L K⁺ pipette solution. Cell capacity was not different between CA patients (22±2 pF) and non–cancer patients (20±4 pF). The Ca²⁺-activated cell current was K⁺ selective as reversal potential shifts depended on the extracellular K⁺ concentration (Figure 2B). In symmetrical K⁺ solutions, the K⁺ current exhibited slight inward rectification in a membrane potential range of -100 to +100 mV. The current was completely blocked by 100 nmol/L charybdotoxin (CTX; n=6, K_d=6.0±0.2 nmol/L), a blocker of hIK1 and hSlo (Figure 2C). Clotrimazole (CLT, 1 µmol/L, n=3, Figure 2D), a more selective blocker of hIK1, reduced the outward current by 90%; K_d=148±22 nmol/L (Figure 2E). In contrast, apamin (100 nmol/L, n=3) had no effect on K⁺ currents, thus indicating that small-conductance K_Ca channels do not considerably contribute to endothelial K_Ca currents in endothelium of MA.

View larger version (24K): [in this window] [in a new window]   Figure 2. Representative whole-cell recordings of ECs from MA of a CA patient. For KCa activation, ECs were dialyzed with 13 µmol/L [Ca2+]free. A, Whole-cell currents recorded immediately after cell membrane rupture (a) and after 15 seconds of cell dialysis (b). B, Ca2+-activated currents after substitution of the 4.5 mmol/L K+ bath solution (a) by a 140 mmol/L K+ solution (b, left); K+ selectivity of ICa (b, right). Each point represents reversal potentials (Erev, n=3 to 6) of ICa at the designated [K+]o measured in ECs with low basal and Ca2+-independent currents. Straight line indicates linear regression to the data (slope 56 mV). C, Inhibition of Ca2+-activated outward currents by CTX. Currents in the absence of CTX (a), in the presence of 100 nmol/L CTX (b), and after washout (c). D, Inhibition of Ca2+- activated outward currents by CLT. Shown are currents in the absence of CLT (a) and in the presence of 1 µmol/L CLT (b). E, Concentration-dependent blockade of KCa currents by CTX and CLT (n=3 to 5; Kd=148±22 nmol/L) determined at +50 mV. Solid lines indicate CTX blockade (n=3 to 6), with cells containing only hIK1 current fraction (Kd=6.0±0.2 nmol/L). Dashed line indicates a cell containing hIK1 and hSlo currents (Kd=6.2 nmol/L). F, Representative whole-cell recordings of an EC dialyzed with a nominally free Ca2+ solution to unmask hSlo currents from hIK1 currents (F). Five current recordings were overlaid.

The K_Ca current in ECs from MAs of both groups matched the properties of hIK1 and showed characteristics of inward rectification, CTX and CLT sensitivity, and Ca²⁺ dependence of channel activation comparable with those of human hIK1 cloned from human pancreas and T cells.^{15 16}

Another type of K⁺ current fraction was detected in a subset of ECs (n=4) from CA patients. Outward currents exhibited voltage sensitivity with a characteristic current activation at depolarizing membrane potentials in the range of +60 to +100 mV (Figure 2D). Despite the small number of observations, these K⁺ currents had hSlo characteristics and showed similarities to whole-cell currents of human hSlo functionally expressed in bovine pulmonary arterial ECs.¹⁷

In inside-out single-channel recordings, we identified hIK1 in both patient groups. In non–cancer patients, hIK1 activity showed no voltage sensitivity and exhibited inward rectification. Mean single-channel conductance of hIK1 was 31±2 pS (n=5), with a conductance of 42±2 pS at negative membrane potentials ranging from -80 to -20 mV and 9±2 pS at positive membrane potentials ranging from +20 to +100 mV (Figure 3A). In excised patches with a 140 mmol/L K⁺ pipette solution and a 4.5 mmol/L K⁺ bath solution, hIK1 was selectively permeable for K⁺ (Figure 3B). The extrapolated reversal potential was +42 mV, corresponding to a K⁺:Na⁺ permeability ratio of 1:0.17, as calculated by the Goldmann-Hodgkin-Katz equation.¹³ Single-channel activity of hIK1 strongly depended on the Ca²⁺ concentration at the cytosolic face of the channel (Figure 3C), with half-maximal activation (EC₅₀) at 352±61 nmol/L as determined at [Ca²⁺]_free ranging from 10 nmol/L to 20 µmol/L (Figure 3D). In CA patients, the hIK1 showed similar characteristics regarding Ca²⁺ dependence (EC₅₀ 370±67 nmol/L) and mean conductance of 29±1 pS (n=8) and of 43±2 and 9±2 pS at negative and positive membrane potentials, respectively. These single-channel properties of hIK1 in ECs from MAs of both groups are comparable with those of human hIK1 cloned from human pancreas and T cells.^{15 16}

View larger version (19K): [in this window] [in a new window]   Figure 3. Single-channel properties of hIK1 and hSlo. Po indicates open probability. A, Inward (down) and outward (up) hIK1 currents in an inside-out patch (140 mmol/L K+ pipette, 140 mmol/L K+ bath, 400 nmol/L [Ca2+]free) from non–cancer patients. B, Current-voltage relationship of hIK1 in symmetrical 140 mmol/L K+ (inside-out, ), 140 mmol/L K+ pipette, and 4.5 mmol/L K+ bath (inside-out, ; outside-out, ). C, Representative traces showing hIK1 currents at cytosolic [Ca2+]free as indicated. Membrane potential was -80 mV. D, Relationship between open probability and cytosolic [Ca2+]free. Points are mean±SE of the designated number of experiments. Data were fitted to the Boltzmann equation. E, Inward (down) and outward (up) hSlo currents in an inside-out patch (symmetrical 140 mmol/L K+ and 400 nmol/L [Ca2+]free) from a CA patient. F, Current-voltage relationship of hSlo in symmetrical 140 mmol/L K+ () and 140 mmol/L K+ pipette, 4.5 mmol/L K+ bath in outside-out (•) and inside-out () patches. G, Trace showing hSlo currents from an inside-out patch containing 2 channels at 0 mV (4.5 mmol/L K+ pipette and 140 mmol/L K+ bath) and at cytosolic [Ca2+]free and with open probabilities as indicated. C denotes closed state of channel.

We estimated the hIK1 channel density per hIK⁺ cell by assuming a single-channel amplitude of 1 pA at 0 mV and an open probability (P_o) of 0.28 and by measuring peak Ca²⁺-activated whole-cell currents at 0 mV to minimize contamination by nonselective and Cl^- currents. On the basis of these assumptions, the estimated hIK1 density in ECs with a hIK1⁺/hSlo^- current pattern was 740 channels per cell in non–cancer patients and 930 channels per cell in CA patients.

Single-channel currents of hSlo were observed in ECs from CA patients. hSlo (n=3) had a mean conductance of 208±16 pS (Figure 3E), exhibited no inward rectification, and showed voltage sensitivity with increasing activity at depolarizing membrane potentials. In excised patches with a 140 mmol/L K⁺ pipette solution and a 4.5 mmol/L K⁺ bath solution, hSlo was selectively permeable for K⁺ (Figure 3F). The extrapolated reversal potential was +58 mV, corresponding to a K⁺:Na⁺ permeability ratio of 1:0.08. hSlo activity was Ca²⁺ dependent with an EC₅₀ of 6 µmol/L cytosolic Ca²⁺ (n=1) at 0 mV (Figure 3G).

We estimated hSlo density in a single EC with hIK1⁺/hSlo⁺ current pattern by assuming a single-channel amplitude of 25 pA and a P_o of 1 at +100 mV and by determining the iberiotoxin (IbTX)–sensitive peak Ca²⁺-activated whole-cell current at +100 mV. On the basis of these assumptions, an estimated hSlo density in hIK1⁺/hSlo⁺ cells of 35 channels per EC was calculated.

hSlo currents were not detected in 105 patches obtained from non–cancer patients. This apparent lack of hSlo currents indicates that hSlo expression is very low or absent in ECs of non–cancer patients.

Subsequent to patch-clamp measurements, single-cell RT-PCR analysis revealed that all of the vWF⁺ cell samples (n=8) with an inwardly rectifying K_Ca current fraction yielded a positive hIK1 PCR signal (Figure 4). In CA patients, hSlo transcripts could be amplified from all ECs exhibiting hSlo-related whole-cell currents (Figure 4). The respective K_Ca current fractions were sensitive to CLT and to IbTX, a selective blocker of hSlo. hIK1 or hSlo transcripts could be not amplified from any of the cell samples (n=14) with no Ca²⁺-activated K⁺ current, although these samples were positive for vWF, indicating successful EC harvesting. On the basis of this parallelism, single-cell RT-PCR analysis apparently differentiates between K_Ca channel–expressing and K_Ca channel–lacking ECs. A heterogeneity of channel expression within a population of ECs of the monolayer and, moreover, a restriction of Ca²⁺-activated K⁺ channel expression to a subset of ECs have not yet been described in ECs. Regarding Ca²⁺ signaling, for example, the generation of Ca²⁺ waves, a functional heterogeneity within ECs, has thus far only been described in lung capillary ECs, which indicated a pacemaker function of some specialized ECs.¹⁸ It is tempting to speculate that the subset of hIK1-expressing ECs might also represent a specialized population of ECs for the control of membrane potential in response to endothelial stimulation.

View larger version (39K): [in this window] [in a new window]   Figure 4. Expression pattern of KCa genes and corresponding whole-cell recordings in a single EC from MA of a CA patient (top lane). Ethidium bromide–stained gel of multiplex RT-PCR products from 5 ECs, 1 -RT control, and 2 medium controls. Shown are corresponding fast whole-cell recordings of 3 ECs (upper traces), as follows: negative for hIK1 mRNA expression (left), positive for hIK1 and negative for hSlo mRNA expression (middle), and positive for hIK1 and hSlo mRNA expression (right). KCa currents of another hIK1+/hSlo- cell (lower traces, middle) before (a) and after application of CLT (100 nmol/L [b]) and another hIK1+/hSlo+ cell (lower lane, right) before (a) and after application of IbTX (100 nmol/L [b]) followed by CLT (100 nmol/L, [c]; 1 µmol/L [d]).

To characterize the functional role of K_Ca channels in the control of membrane potential in both groups, we performed current-clamp experiments in each vessel preparation and determined resting potentials and agonist-induced hyperpolarization in the electrically coupled ECs of the intact monolayer. Application of bradykinin (100 nmol/L) induced sustained hyperpolarization of the membrane potential (Figure 5A). In the presence of 100 nmol/L CTX (n=9), but not 100 nmol/L apamin, a blocker of small-conductance K_Ca channels (n=5), membrane potential shifts were completely reversed, thus confirming the activity of CTX-sensitive K_Ca channels (Figure 5B). To discriminate the contribution of hIK1 and hSlo to bradykinin-induced hyperpolarization of mesenteric endothelium, we determined the blocking effects of CLT (n=4) and IbTX (n=4). Application of CLT (1 µmol/L) shifted to the same extent as CTX the membrane potential back to the resting potential (Figure 5C). In contrast, application of IbTX (100 nmol/L, Figure 5D) had no detectable effect on bradykinin-induced hyperpolarization, although hSlo expression was detected in the respective ECs from CA patients. This indicated that activation of mainly hIK1 mediates endothelial hyperpolarization in response to bradykinin in both patient groups. Activation of hSlo seems to play a minor role or no role in bradykinin-induced hyperpolarization of mesenteric endothelium from CA patients.

View larger version (12K): [in this window] [in a new window]   Figure 5. Current-clamp recordings of the endothelial membrane potential of an MA from a CA patient. Endothelial hyperpolarization was induced by applying 100 nmol/L bradykinin (BK [A]). Inhibition of hyperpolarization by 100 nmol/L CTX (B) and by 1 µmol/L CLT (C) but not by 100 nmol/L IbTX (D). The individual ECs were hIK1+/hSlo+. E, Percentage of hIK1-positive ECs correlates with the degree of hyperpolarization. P<0.01, Spearman rank correlation.

To relate electrophysiological and RT-PCR data, we sought to determine whether the percentage of hIK1 expressing ECs per vessel correlates with the ability of the endothelium to hyperpolarize in response to bradykinin. The degree of bradykinin-induced hyperpolarization correlated significantly with the percentage of hIK1-positive cells collected from the respective vessel preparations (Figure 5E). This finding supports the idea of a predominant role of hIK1-expressing ECs in the control of agonist-induced hyperpolarization.

Comparison of Endothelial K_Ca Channel Expression in MA From Cancer and Non–Cancer Patients
To demonstrate the usefulness of our technical approach, we tested whether alterations of K_Ca channel expression and function occur in disease states. Ca²⁺ homeostasis and K⁺ channel function in the endothelium have been suggested to play a role in the regulation of angiogenesis.^{10 11} We therefore compared expression of hIK1 and hSlo in single ECs of MA specimens obtained from CA patients (n=8) and from non–cancer patients (n=7). To compare K_Ca channel expression in endothelium, 10 to 15 ECs were harvested from MA preparations of each patient, and the percentage of K_Ca-positive ECs was determined.

The percentage of hIK1-positive ECs per vessel was significantly increased in MAs from CA patients compared with controls (P<0.05; Figure 6A). As mentioned above, hSlo-positive ECs were exclusively detected in MAs from CA patients (Figure 6B). Interestingly, hSlo was exclusively detected in hIK1-positive cell samples. This coexpression might indicate that hSlo expression is coupled to hIK1 expression in ECs from CA patients.

View larger version (19K): [in this window] [in a new window]   Figure 6. Expression of hIK1 and hSlo in endothelium of MA from CA patients (n=8) and non–cancer patients (CON, n=7). Expression of hIK1 (A) and hSlo (B) was determined in each vessel as a percentage of ECs yielding a hIK1- or hSlo-positive RT-PCR signal. C, Bradykinin-induced shift of membrane potential (Vm) in endothelium of MA from CA and CON. Values are mean±SE. n.d. indicates not detectable. *P<0.05, Mann-Whitney U test.

To determine whether increased percentage of K_Ca channel-expressing ECs leads to an increased ability of the endothelium of CA patients to hyperpolarize in response to agonist-induced Ca²⁺ release from internal stores, we compared bradykinin-induced hyperpolarization in endothelium of MAs from each group. Resting potentials did not statistically differ between CA patients (-32±5 mV) and controls (-28±5 mV, NS). The degree of hyperpolarization in response to 100 nmol/L bradykinin was significantly increased in CA patients (shift of membrane potential [V_m], -22±4 mV) compared with controls (V_m, -8±3 mV, P<0.05; Figure 6C).

These data suggest that, compared with non–cancer patients, increased expression of hIK1 in endothelium of MA from CA patients leads to considerably increased hyperpolarization in response to agonist stimulation. The physiological role of hSlo expression, which did not considerably contribute to endothelial hyperpolarization in CA patients, remains unclear.

Given that hIK1-mediated hyperpolarization increases the electrochemical driving force for Ca²⁺ influx, our findings may indicate an altered control of endothelial function and Ca²⁺ signaling in endothelium of MA from CA patients. hIK1 expression has been shown to regulate mitogenic cell growth and proliferation in other tissues, such as human fibroblasts,¹⁹ and in activated T lymphocytes.¹⁶ However, the question of whether the increased expression of hIK1 in endothelium of CA patients is indicative of mitogenic cell growth and angiogenesis requires further studies to resolve.

In conclusion, we newly established combined single-cell RT-PCR and patch-clamp analyses in human blood vessels. This allows functional and molecular biological characterization of single ECs in situ by avoiding alterations due to cell isolation and culture. We directly correlated expression and function of the K_Ca channels, hIK1 and hSlo, at the single-cell level. The heterogeneous expression patterns of hIK1 are indicative of a population of specialized ECs within the endothelium. A disease-related alteration in the expression pattern of K_Ca channels in cancer patients could be involved in endothelial function during angiogenesis.

	Acknowledgments

 
This work was supported by the Deutsche Forschungsgemeinschaft (FOR 341/1, FOR 341/5, and Ho 1103/2-4).

	Footnotes

 
Reprint requests to J. Hoyer, Benjamin Franklin Medical Center, Hindenburgdamm 30, 12200 Berlin, Germany.

Received April 4, 2000; revision received July 19, 2000; accepted July 20, 2000.

	References

Top Abstract Introduction Materials and Methods Results and Discussion References

 
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