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Keywords: 0335 Journal of Physiology (2000), 525.2, pp. 307—317 307 ATP-sensitive potassium channels in capillaries isolated from guinea-pig heart Michael Mederos y Schnitzler, Christian Derst, Jurgen Daut and Regina Preisig_Muller Institut fur Normale und Pathologische Physiologie, Universitat Marburg, Deutschhausstrasse 2, D_35037 Marburg, Germany (Received 17 November 1999; accepted after revision 13 March 2000) 1. The full-length cDNAs of two different á_subunits (Kir6.1 and Kir6.2) and partial cDNAs of three different â_subunits (SUR1, SUR2A and SUR2B) of ATP-sensitive potassium (KATP) channels of the guinea-pig (gp) were obtained by screening a cDNA library from the ventricle of guinea-pig heart. 2. Cell-specific reverse-transcriptase PCR with gene-specific intron-spanning primers showed that gpKir6.1, gpKir6.2 and gpSUR2B were expressed in a purified fraction of capillary endothelial cells. In cardiomyocytes, gpKir6.1, gpKir6.2, gpSUR1 and gpSUR2A were detected. 3. Patch-clamp measurements were carried out in isolated capillary fragments consisting of 3—15 endothelial cells. The membrane capacitance measured in the whole-cell mode was 19·9 ± 1·0 pF and was independent of the length of the capillary fragment, which suggests that the endothelial cells were not electrically coupled under our experimental conditions. 4. The perforated-patch technique was used to measure the steady-state current—voltage relation of capillary endothelial cells. Application of K¤ channel openers (rilmakalim or diazoxide) or metabolic inhibition (250 ìÒ 2,4-dinitrophenol plus 10 mÒ deoxyglucose) induced a current that reversed near the calculated K¤ equilibrium potential. 5. Rilmakalim (1 ìÒ), diazoxide (300 ìÒ) and metabolic inhibition increased the slope conductance measured at −55 mV by a factor of 9·0 (±1·8), 2·5 (±0·2) and 3·9 (±1·7), respectively. The effects were reversed by glibenclamide (1 ìÒ). 6. Our results suggest that capillary endothelial cells from guinea-pig heart express KATP channels composed of SUR2B and Kir6.1 andÏor Kir6.2 subunits. The hyperpolarization elicited by the opening of KATP channels may lead to an increase in free cytosolic Ca¥, and thus modulate the synthesis of NO and the permeability of the capillary wall. Endothelial cells show complex changes in membrane potential upon application of vasoactive agonists (Mehrke & Daut, 1990; Marchenko & Sage, 1993; McGahren et al. 1998; Frieden et al. 1999) and are able to produce membrane potential oscillations (Usachev et al. 1995). Many of the functions of the endothelium, for example, the release of vasoactive compounds and the regulation of the permeability of the vascular wall, are influenced by the free intracellular calcium concentration, which increases with hyperpolarization (Cannell & Sage, 1989) and sometimes shows pronounced oscillations (Jacob, 1991; Usachev et al. 1995; Langheinrich et al. 1998). Thus the electrical activity of the endothelial cells can have profound effects on endothelial function. Yet the role of different ion channels in generating the electrical responses of the endothelium is still far from clear. In the present study we focus on the structure and function of ATP-sensitive potassium channels (KATP channels) in microvascular endothelium. KATP channels are octamers (Shyng & Nichols, 1997; Clement et al. 1997) composed of four á_subunits (inward rectifier channels of the Kir6 subfamily, Kir6.1 or Kir6.2; Inagaki et al. 1995b; Sakura et al. 1995) and four â_subunits (sulfonylurea receptors, SUR1 or SUR2; Aguilar-Bryan et al. 1995; Inagaki et al. 1996; Isomoto et al. 1996). The channels are characterized by the dependence of their open-state probability on the concentrations of intracellular ATP, ADP and other nucleotides (Yamada et al. 1997; Trapp et al. 1998; Gribble et al. 1998). KATP channels can be activated pharmacologically by a chemically heterogeneous class of compounds designated K¤ channel openers and can be blocked by sulfonylurea derivatives. Furthermore, the open- 308 M. Mederos y Schnitzler, C. Derst, J. Daut and R. Preisig-Muller state probability of KATP channels can be modulated by vasoactive substances such as adenosine, calcitonin generelated peptide and angiotensin II (Dart & Standen, 1993; Quayle et al. 1994; Kubo et al. 1997) and by numerous intracellular factors such as pH, lactate, protein kinase A and protein kinase C (Han et al. 1993; Kleppisch & Nelson, 1995; Bonev & Nelson, 1996; Baukrowitz et al. 1999). KATP channels have been found in many different cell types including cardiac and skeletal muscle cells, arterial smooth muscle cells, pancreatic â_cells and some neuronal cells. In the endothelium, the existence of KATP channels is still controversial. Most of our present knowledge on the electrophysiology of vascular endothelial cells has been derived from studies on cultured macrovascular endothelial cells (Nilius et al. 1997), where KATP channels are usually not observed. On the other hand, some evidence for the existence of KATP channels has been obtained in primary cultures of microvascular endothelial cells from rat brain (Janigro et al. 1993), in freshly isolated aortic endothelial cells (Katnik & Adams, 1995, 1997) and in freshly isolated coronary endothelial cells (Langheinrich & Daut, 1997). Here we report a study of the expression of á- and â_subunits of KATP channels in capillary endothelial cells isolated from guinea-pig heart using cell-specific RT-PCR. In addition, we have carried out patch-clamp measurements in capillary fragments from guinea-pig heart. We have found a potassium current that can be activated by K¤ channel openers and blocked by glibenclamide. Taken together, our results suggest that capillary endothelial cells from guinea-pig heart express KATP channels consisting of the á_subunits Kir6.1 andÏor Kir6.2 and the â_subunit SUR2B. Preliminary reports of some of our findings have been published (Mederos y Schnitzler et al. 1998, 1999; Preisig-Muller et al. 1999a,b). METHODS Guinea-pigs weighing 200—380 g were killed without prior anaesthesia by decapitation with a small-animal guillotine, in accordance with the guidelines laid down by the regional animal care committee (at the Regierungsprasidium, Giessen, Germany). The heart was quickly excised and the aorta attached to a perfusion cannula. Subsequently, coronary capillaries and ventricular myocytes were isolated by enzymatic digestion. Cloning of KATP channel subunits from guinea-pig heart A standard reverse transcription-polymerase chain reaction (RTPCR) method was used to amplify SUR1, SUR2 and Kir6.2 cDNA fragments from guinea-pig (gp) heart RNA. Degenerate primers based on the carboxyterminal amino acid sequence of the different SUR isoforms were used to amplify the SUR cDNA fragments: SUR1, amino acids 1395—1402 (forward) and 1556—1563 (reverse) of rat SUR1 (GenBank accession number L40624); SUR2, amino acids 1321—1329 (forward) and 1454—1460 (reverse) of rat SUR2 (accession number D83598). Degenerate primers based on amino acids 79—86 (forward) and 336—343 (reverse) of rat Kir6.2 primer (accession number U44897) were used to amplify the Kir6.2 cDNA fragment. The amplified cDNA fragments were cloned into pBluescript SK+ vector (Stratagene) and sequenced using an J. Physiol. 525.2 automatic nucleotide sequencer (Genetic Analyser 310, Applied Biosystems, Foster City, CA, USA). To obtain a Kir6.1 DNA probe, an 800 bp fragment from a human Expressed Sequence Tag clone (GenBank accession number N70558, IMAGp998H19669, kindly supplied by the Resource Centre of the German Human Genome Project, Max-Planck-Institute for Molecular Genetics, Berlin, Germany) was isolated after digestion with Not I and Eco RI. All Kir6 and SUR cDNA fragments were isolated and nonradioactively labelled with digoxigenin-11dUTP (Boehringer, Mannheim, Germany) for screening cDNA or genomic libraries. A commercial kit (Great Lengths cDNA Synthesis Kit, Clontech) was used to construct a cDNA library from 5 ìg heart ventricle poly(A)¤ RNA. The adaptor-ligated cDNA was inserted into the ëTriplEx vector (Clontech) and plated on the Escherichia coli strain XL1-blue. Subsequently, 1 ² 10É independent plaques were screened with the non-radioactively labelled cDNA probes using standard plaque hybridization and chemiluminescence detection protocols (Boehringer). Clones that hybridized to the DNA probes were isolated and re-screened to obtain single positive clonal plaques. The isolated single phage clones were further characterized by converting the phage DNA, rescuing the respective pTriplEx plasmids and analysing their cDNA inserts by restriction mapping and sequencing. Genomic gpKir6.1 and gpKir6.2 clones were isolated from a guinea-pig FIX II genomic library (Stratagene) plated on the E. coli strain XL1-blue MRA(P2); 1 ² 10É independent phages were screened with the specific Kir6.1 and Kir6.2 probes using protocols described above. The ë DNAs of single phage clones were prepared using the ë-Midi Kit (Qiagen, Hilden, Germany) and digested with different restriction enzymes to produce further subclones in the pBluescript SK+ vector. Total sequence information was obtained by either sequencing these subclones or by direct sequencing of ë DNA. All subclonings were performed using standard cloning techniques (Sambrook et al. 1989). Isolation of capillary fragments and cardiac muscle cells for RT-PCR Capillary fragments and cardiomyocytes were isolated as previously described (Preisig-Muller et al. 1999c). Briefly, a mixed cell suspension from guinea-pig heart was obtained by collagenase digestion, and capillary fragments or cardiomyocytes were enriched using sieving techniques and gravity sedimentation, respectively. For cell picking, several drops of the enriched cell suspension were transferred to 35 mm Petri dishes filled with physiological salt solution to which 1% BSA had been added to prevent attachment of the cells. The Petri dish with the diluted cell suspension was mounted on an inverted microscope (Zeiss IM 35). Capillary fragments consisting of 6—15 endothelial cells or single cardiomyocytes were collected under visual control with a hydraulic cell picker. The purity of the cell fractions was verified by multicell RT-PCR experiments using the cell-specific markers endothelin-1 and troponin T. RNA extraction, reverse transcription and polymerase chain reaction Total RNA from different tissues was isolated using a modified acid guanidinium—phenol method (Chomczynski & Sacchi, 1987). Total RNA from pure cell fractions containing about 1000 cardiomyocytes or 150 capillary fragments was prepared using a commercial kit (RNeasy Mini Kit, Qiagen) according to the manufacturer’s instructions. First-strand cDNA was synthesized from 2 ìg tissue RNA or one-third of the cell RNA eluate using 200 units of Superscript II reverse transcriptase (Gibco BRL) and a random hexanucleotide mixture, or an anchor-oligo(dT) (5'-GTC- J. Physiol. 525.2 ATGGCATGGGATCCTG(T)15; total volume, 25 ìl). The PCR was performed with gene-specific and intron-spanning primers to avoid a possible amplification of genomic DNA. The 3'-RACE (rapid amplification of cDNA ends) method was used in the case of the intronless Kir6.2 gene. The total PCR volume was 50 ìl, including 1Ï12 of the RT reaction, 50 pmol of each primer and 2·5 units AmpliTaq Gold (Applied Biosystems). PCR was performed in a thermal cycler Model 2400 (Applied Biosystems) under the following conditions: the PCR was run with a hot-start for 5 min at 94°C (initial melt), then for 40 cycles of 0·5 min at 94°C, 0·5 min at 55°C and 1 min at 72°C and, finally, for 5 min at 72°C (final extension). The primers had the following sequences. For gpKir6.1: sense, 5'-GTCCTTCCTCTGCAGTTGGC-3'; antisense, 5'-CATGA CAGCGTTGATGATCAGACC-3'. For gpKir6.2: sense, 5'-GACAAG CTGAGTAGAGAGACTGAGG-3'; antisense primers annealing to the anchor-oligo(dT), 5'-GTCATGGCATGGGATCCTG(T)×. For gpSUR1: sense, 5'-CATGGTGGACATGTTCGAGGGCCGC-3'; antisense, 5'-CTGGTTTGTCGAACTCCAGGATGGC-3'. For gpSUR2A and gpSUR2B: sense 5'-GTTGACATATTTGATGGAA AG-3'; SUR 2A antisense 5'-CTACTTGTGAGTCATCACCAAGGT_3'; SUR2B antisense, 5'-GCACGAACAAAAGAAGCAAAT-3'. Isolation of capillaries for patch-clamp experiments The coronary arteries of the isolated heart were perfused at a constant flow rate of 6—9 ml min¢ using a peristaltic pump. The heart was submerged in a small organ bath warmed to 37°C and coronary perfusion pressure (CPP) was measured with a pressure transducer (PMT, Ettringen, Germany). Initially the hearts were perfused with physiological salt solution containing (mÒ): 135 NaCl, 5 KCl, 1 CaClµ, 1 MgClµ, 0·33 NaHµPOÚ, 2 sodium pyruvate, 10 glucose and 10 Hepes. The pH was 7·4 (adjusted with NaOH); the temperature was 37°C. When the CPP had recovered to about 80 mmHg the heart was arrested with an otherwise identical solution containing 15 mÒ K¤ and 125 mÒ Na¤. Within 10 min this caused a further increase in CPP to about 90 mmHg, indicating recovery of energy metabolism. In order to initiate dissociation of the cells, the heart was perfused for 5 min with nominally Ca¥-free solution, which otherwise had the same composition as described above (15 mÒ K¤). Then the heart was perfused for 10 min with Ca¥-free solution to which 30 ìÒ Ca¥ and 1·5 mg ml¢ of collagenase blend, Type H (Sigma), or collagenase Type 2 (CLS-2, Worthington) were added. Subsequently, the heart was removed from the organ bath and placed in a ‘storage’ solution containing (mÒ): 65 potassium glutamate, 45 KCl, 30 KHµPOÚ, 3 MgSOÚ, 0·5 EGTA, 20 taurine and 10 glucose (pH adjusted to 7·4 with KOH; temperature, 22°C). The ventricles were cut into small pieces and disintegrated by trituration with a wide-tipped Pasteur pipette. Drops of the suspension were transferred immediately to 35 mm Petri dishes (Nunc, Denmark) containing the same solution. After 20 min, nonadhering cells were washed away with physiological salt solution at room temperature. Usually several capillary fragments remained attached to the bottom of the Petri dishes. At least 30 min before the start of electrophysiological recording, 350—400 units of deoxyribonuclease (DNAse I, Type IV, Sigma) were added to each Petri dish to clean the surface of the cells. Electrophysiology, solutions and reagents 309 KATP channels in capillaries Patch-clamp recordings were carried out on the stage of an inverted microscope (Olympus IX 70) at room temperature (23°C) within 12 h after seeding the suspension on the Petri dishes. After selecting a capillary fragment adhering to the bottom of a Petri dish, a Perspex frame was mounted over the capillary, as described previously (Daut et al. 1988). The frame formed a perfusion chamber of 1 mm height, 1·5 mm width and 22 mm length, which was perfused with normal physiological salt solution at a rate of 5—10 ml h¢. In some experiments the K¤ concentration of the superfusing solution was elevated to 140 mÒ. In this solution Ca¥ was omitted, and Na¤ was reduced to keep osmolarity constant. Voltage-clamp experiments were carried out both in the conventional whole-cell mode of the patch-clamp technique (n = 28) and in the perforated-patch mode (n = 31). For perforated-patch measurements, pipettes of 1—2 ìm tip diameter were made of thinwalled glass of 1·5 mm diameter without filament (Science Products, Hofheim, Germany). They were coated with Sylgard to reduce capacitance and heat-polished directly before use. The resistance of the pipettes was 5—8 MÙ. The pipette solution for perforated-patch measurements contained (mÒ): 45 KCl, 100 potassium aspartate, 1 MgClµ, 0·5 EGTA and 10 Hepes (pH 7·2, adjusted with NaOH). With this solution, Donnan potentials at the perforated patch were assumed to be negligible. The tip of the patch electrode was first filled with amphotericin-free pipette solution (column length, 400—500 ìm) by aspiration, then the pipette was backfilled with the same solution to which 300 ìg ml¢ amphotericin B had been added from a stock solution. Sonication was applied to improve solvation of amphotericin B. The stock solution contained 20 mg ml¢ amphotericin B in DMSO and was freshly prepared each day. Perforation started shortly after seal formation and reached a steady-state level within 5—10 min. The pipette solution used for conventional whole-cell recordings contained (mÒ): 45 KCl, 100 potassium aspartate, 10 EGTA, 1 CaClµ, 3 MgClµ, 2 NaµATP, 0·1 Na×GTP and 10 Hepes (pH 7·2, adjusted with NaOH). After forming a gigaohm seal and compensating electrode capacitance, the patch membrane was ruptured by application of suction. The membrane capacitance was calculated from the capacitive current offsets elicited by voltage ramps with a slope of 0·5 V s¢. Recordings were carried out initially with an EPC_7 patch-clamp amplifier (List, Germany) and a modified digital audiotape recorder (Sony, DTC-55ES; sampling rate, 44 kHz). In later experiments, an Axopatch 200B amplifier (Axon Instruments) was used. In all whole-cell recordings, membrane capacitance and series resistance (50%) were compensated. The data were filtered with a cut-off frequency that was half the sampling rate, which was in the range 500—5000 Hz. The measured membrane potentials were corrected for the liquid junction potential determined as described by Neher (1992). In the perforated-patch measurements, for example, the liquid junction potential was +8 mV with physiological salt solution (5 mÒ K¤) and +3·7 mV with high-potassium solution (140 mÒ K¤). Where appropriate, the data are given as means ± standard error of the mean (s.e.m.); n denotes the number of capillaries from which the data were obtained. RESULTS Cloning of KATP channel subunits from guinea-pig heart KATP channels are composed of inward rectifier channels of the Kir6 subfamily (á-subunits) and sulfonylurea receptors (â-subunits). To obtain guinea-pig specific sequences, all subunits were isolated from a cardiac ventricular cDNA library. Full length cDNAs of gpKir6.1 (2066 bp, GenBank accession number AF183918) and gpKir6.2 (2762 bp, accession number AF183919) with the entire non-coding regions were obtained. The nucleotide sequences of the gpKir6.1 and gpKir6.2 cDNAs predict single open-reading 310 M. Mederos y Schnitzler, C. Derst, J. Daut and R. Preisig-Muller frames of 424 and 390 amino acids, respectively. Comparison of the deduced gpKir6.1 and gpKir6.2 amino acid sequences with the corresponding human Kir6 amino acid sequences showed 98·6 and 96·2% identity, respectively (hKir6.1: accession number D50312, Inagaki et al. 1995b; hKir6.2: accession number D50582, Inagaki et al. 1995a). Partial clones of gpSUR1 (1663 bp, accession number AF183921), gpSUR2A (3052 bp, accession number AF183922) and gpSUR2B (3576 bp, accession number AF183923) were obtained, which encode the carboxyterminal 331, 423 and 319 amino acids, respectively. Genomic clones of gpKir6.1 (KCNJ8; 3480 bp, accession number AF196330, partial clone) and gpKir6.2 (KCNJ11; 4589 bp, accession number AF183920, complete clone) were isolated from a genomic library of the guinea-pig. The Kir6.1 partial clone started with an intronic sequence followed by exon 3 and the complete 3' non-coding region. A comparison of cDNA and genomic clones showed that one intron splits the coding region of the gpKir6.1 gene (at position 456 bp in the cDNA clone) and that the gpKir6.2 gene has no intron. J. Physiol. 525.2 Tissue- and cell-specific distribution of KATP channel subunit transcripts Gene-specific primers for Kir6.1, Kir6.2, SUR1, SUR2A and SUR2B were designed as described in Methods. The sensitivity and the selectivity of the primers was tested with total RNA isolated from different organs. Figure 1 shows the results of RT-PCR experiments using 120 ng total RNA as template. The size of the amplified PCR products was as expected from the cloned sequences. The specificity of the amplified DNA fragments was verified by direct sequencing. Transcripts of all KATP channel subunits investigated were found in atria and ventricles of guinea-pig heart. The gene expression of SUR2A was found to be restricted to heart, skin and skeletal muscle. Kir6.2 was found to be strongly expressed in the same tissues as SUR2A and, in addition, at a low level in all organs examined. The Kir6.1, SUR2B and SUR1 genes were found to be ubiquitously expressed in guinea-pig. Approximately equal amounts of total RNA were used for reverse transcription, as can be seen from the RT-PCR lanes for glyceraldehyde-3phosphate dehydrogenase (GAPDH) in Fig. 1. Figure 1. Tissue distribution of Kir6 and SUR transcripts in guinea-pig RT-PCR of gpSUR1, gpSUR2A, gpSUR2B, gpKir6.1, gpKir6.2 and gpGAPDH in different tissues. Representative 2% agarose gels were loaded with one-third of each probe and stained with ethidium bromide. To verify the identity of the PCR products, DNA fragments were isolated and directly sequenced. Note that SUR2B-specific primers amplified an additional longer (175 bp) fragment in tissues expressing SUR2A. This can be explained by the fact that, in some of our clones, SUR2A cDNA contained in addition the SUR2B-specific exon 40 in the 3' non-coding region. bp, base pair. J. Physiol. 525.2 KATP channels in capillaries Cell-specific expression of KATP channels in microvascular endothelial cells was studied using the multicell RT-PCR method described previously (Preisig-Muller et al. 1999c). gpKir6.1, gpKir6.2 and gpSUR2B were found to be expressed in capillary fragments isolated from cardiac ventricles, as illustrated in Fig. 2B (upper panel). Endothelin-1 was used as a positive control and specific marker for endothelial cells. Troponin T expression was not found in the capillary fragments, which rules out contamination by cardiomyocytes. The lower panel of Fig. 2B illustrates that gpKir6.1, gpKir6.2, gpSUR1 and gpSUR2A were expressed in cardiomyocytes. Troponin T was used as a positive control and specific marker for cardiomyocytes. Endothelin-1 expression was not found in the cardiomyocytes, which rules out contamination by endothelial cells. Passive electrical properties of isolated capillary fragments The patch-clamp technique was used to study the function of KATP channels in capillary fragments isolated from guinea-pig heart. Gigaohm seal formation in capillary fragments is difficult because the sheet of endothelial cells 311 forming the capillary wall is extremely thin (< 1 ìm) and the basement membrane surrounding the cells is only partially removed during the isolation procedure. To obtain some information about the passive electrical properties of the cells we carried out capacitance measurements in the conventional whole-cell mode. A typical measurement is illustrated in Fig. 3A. Ramp-shaped voltage commands were applied between −30 and −50 mV and the magnitude of the current jumps resulting from the change in dVÏdt was measured. The mean capacitance of the cells was 19·9 ± 1·0 pF (n = 28). The mean input resistance determined from these measurements was 3·30 ± 0·66 GÙ (n = 28). Since the mean seal resistance was only about 18_fold larger (60 ± 12 GÙ), the measured input resistance represents a lower limit of the true input resistance of the cells. We used capillary fragments consisting of 3—15 endothelial cells as determined by counting the nuclei (Fig. 2A). Surprisingly, the measured capacitance was not correlated with the length of the capillary (correlation coefficient, 0·013; not illustrated). This finding suggests that under the prevailing experimental conditions the endothelial cells of Figure 2. Cell-specific expression of Kir6 and SUR transcripts in guinea-pig heart A, photomicrograph of a capillary fragment consisting of five cells. The calibration bar is 50 ìm. The positions of the nuclei are indicated by arrows. B, multicell RT-PCR of gpSUR1, gpSUR2A, gpSUR2B, gpKir6.1 and gpKir6.2 in freshly isolated capillary fragments and cardiomyocytes selected with the ‘cell picker’. Representative 2% agarose gels were loaded with one-third of each PCR probe and stained with ethidium bromide. Endothelin-1 (ET-1) and troponin T (TropT) were used as specific markers of endothelial cells and cardiac muscle cells, respectively (Preisig-Muller et al. 1999c). 312 M. Mederos y Schnitzler, C. Derst, J. Daut and R. Preisig-Muller the capillary fragments were not electrically coupled. Probably the measured capacitance represents the membrane properties of the endothelial cell to which the patch pipette was attached (see Discussion). All subsequent measurements were carried out in the perforated-patch mode to prevent changes in the intracellular milieu. Figure 3B shows a typical membrane potential recording (current clamp) from a capillary fragment. In physiological salt solution containing the normal extracellular K¤ concentration (5 mÒ) the membrane potential fluctuated between −35 and −60 mV. The mean resting potential determined in the perforated-patch mode was −36·6 ± 2·8 mV (n = 31). When the external K¤ concentration was increased to 140 mÒ the membrane potential decreased to about 0 mV and the fluctuations J. Physiol. 525.2 disappeared, which was probably attributable to the decrease in the input resistance of the cells and to a decrease in the driving force for K¤ ions. Figure 3C shows the corresponding steady-state current— voltage relations obtained with slow voltage ramps (40 mV s¢). With 5 mÒ external K¤, the curve showed outward rectification in the range −60 to +35 mV. The mean slope conductance at −55 mV was 322 ± 50 pS (n = 31). The low slope conductance was probably responsible for the marked membrane potential fluctuations observed in the current-clamp mode, which may be related to the opening of single channels. With 140 mÒ external K¤, a slope conductance of 1446 ± 651 pS was found at −55 mV (n = 9) and the reversal potential was near 0 mV (range, −2 to +3 mV). Figure 3. The electrical properties of isolated capillary fragments A, typical whole-cell measurement of membrane capacitance using voltage ramps of alternating polarity with a slope of 0·5 V s¢. B, typical perforated-patch measurement of the membrane potential. The gaps in the record indicate the times at which the voltage ramps shown in C were applied. C, steady-state current—voltage relations with 5 mÒ (upper curve) and 140 mÒ (lower curve) external K¤. The slope of the voltage ramps was 40 mV s¢. J. Physiol. 525.2 KATP channels in capillaries The effects of K¤ channel openers and metabolic inhibition Figure 4A shows a typical record of the effects of the K¤ channel opener rilmakalim on the membrane potential of a capillary fragment. The hyperpolarization induced by rilmakalim was associated with a decrease in the amplitude of the noise. The mean membrane potential measured was −38·0 ± 3·1 mV under control conditions and −73·4 ± 1·7 mV (n = 9) in the presence of 1 ìÒ rilmakalim. The effects of rilmakalim on the steady-state current—voltage relation are illustrated in Fig. 4B. Application of 1 ìÒ rilmakalim induced an outward current at potentials positive to −85 mV and an inward current at more negative potentials. The mean reversal potential was −82·9 ± 1·9 mV (n = 9), which is close to the calculated equilibrium potential of a K¤-selective channel. The difference current (Fig. 4C) was approximately linear in the range −100 to −40 mV but showed a plateau, and a substantial increase in noise, at positive potentials. The effect of rilmakalim could be completely reversed by addition of 1 ìÒ glibenclamide in the continuous presence of the K¤ channel opener (n = 5). These findings suggest that capillary endothelial cells from guinea-pig heart possess KATP channels. 313 The effects of rilmakalim on the slope conductance are summarized in Fig. 5. We compared the slope conductance measured before application of the K¤ channel opener with that measured 3 min after application of the drug in the same preparation. On average, 1 ìÒ rilmakalim increased the slope conductance at −55 mV by a factor of 9·0 ± 1·8. This was close to the maximum effect obtainable with rilmakalim; with higher drug concentrations the change in slope conductance was similar. To obtain some information on the possible subtype of KATP channels expressed we also applied the K¤ channel opener diazoxide, which preferentially acts on the pancreatic-type KATP channel (composed of SUR1 and Kir 6.2). The effects of diazoxide were much smaller than the effects of 1 ìÒ rilmakalim. During application of 300 ìÒ diazoxide the slope conductance at −55 mV increased by a factor of 2·5 ± 0·2 (n = 10) within 3 min. Application of 300 nÒ diazoxide had no measurable effect (n = 3). KATP channels are opened by a decrease in submembrane ATP concentration and an increase in submembrane ADP concentration and are thus sensitive to metabolic inhibition. Inhibition of ATP synthesis was induced by switching to a solution containing 250 ìÒ dinitrophenol (DNP), a mitochondrial uncoupler, and 10 mÒ deoxyglucose in place Figure 4. The effects of rilmakalim and glibenclamide on membrane potential and current— voltage relations A, perforated-patch measurement of the effects of 500 nÒ rilmakalim and 1 ìÒ glibenclamide on the membrane potential of a capillary fragment. B, steady-state current—voltage relations obtained from a different capillary fragment under control conditions (1), during application of 1 ìÒ rilmakalim (0) and in the presence of 1 ìÒ rilmakalim plus 1 ìÒ glibenclamide (8). The arrow indicates the calculated K¤ equilibrium potential (EK). C, the difference current obtained by subtraction of the control curve from the curve measured in the presence of 1 ìÒ rilmakalim. 314 M. Mederos y Schnitzler, C. Derst, J. Daut and R. Preisig-Muller of glucose. Figure 6 shows that the effects of metabolic inhibition on the steady-state current—voltage relation were very similar to those of the K¤ channel openers. The slope conductance at −55 mV was increased during metabolic inhibition by a factor of 3·9 ± 1·7 (n = 4) within 3 min. These findings support the idea that functional KATP channels are expressed in capillary endothelial cells from guinea-pig heart. DISCUSSION Patch-clamp recording from capillary fragments Patch-clamp and RT-PCR experiments were done on freshly isolated capillaries to avoid changes in gene expression induced by cell culture. The distinct morphology of coronary capillaries allows visual identification of endothelial cells under the microscope. Capillary fragments containing 3—15 endothelial cells were used for conventional whole-cell recording. After placement of the patch pipette on one of the cells near the nuclear region, compensation of electrode capacitance and rupture of the cell membrane, a mean capacitance of 20 pF was found. We think that this represents the capacitance of single endothelial cells, for the following reasons. (1) The measured capacitance was not J. Physiol. 525.2 correlated with the number of the cells in the capillary fragment. (2) Assuming a specific membrane capacitance of 1 ìF cm¦Â, the mean capacitance observed corresponds to a membrane surface area of 20 ² 10¦É cmÂ. The membrane surface area of a single endothelial cell forming a smooth double cylinder of 30 ìm length and 6 ìm diameter is estimated to be about 11·3 ² 10¦É cmÂ. This leaves a factor of 1·8 for the enlargement of the endothelial cell surface by caveolae, which is consistent with morphological measurements (McGuire & Twietmeyer, 1983). These findings suggest that the endothelial cells were not coupled electrically under our experimental conditions and that the measured currents reflect the electrical properties of single endothelial cells. This need not necessarily reflect the state of the cells in vivo, because we exposed the cells to a Ca¥-free storage solution for at least 30 min, which may lead to uncoupling of the gap junctions. KATP channels in capillary endothelial cells The current induced in capillary endothelial cells by the K¤ channel openers rilmakalim and diazoxide showed an almost linear voltage dependence in the physiological range of potentials (Fig. 4C). This is similar to that found for ATPsensitive potassium channels (Spruce et al. 1987) and other weakly rectifying channels in the presence of physiological external K¤ concentrations. Metabolic inhibition induced a current change that was qualitatively similar to that observed in the presence of the K¤ channel openers rilmakalim and diazoxide. The reversal potential was near the calculated K¤ equilibrium potential. The effects of K¤ channel openers and metabolic inhibition could be reversed by application of 1 ìÒ glibenclamide. These findings suggest that capillary endothelial cells isolated from guinea-pig heart express KATP channels. Using cell-specific RT-PCR, we found that two different á_subunits of KATP channels (Kir6.1 and Kir6.2) were Figure 5. The relative change in slope conductance induced by K¤ channel openers and metabolic inhibition The slope conductance at −55 mV was measured first under control conditions (5 mÒ K¤) and subsequently in the presence of 140 mÒ K¤, 1 ìÒ rilmakalim, 300 ìÒ diazoxide or 250 ìÒ DNP plus 10 mÒ deoxyglucose (metabolic inhibition). The relative increase in slope conductance compared with control in the same capillary fragment is plotted. The number of experiments is indicated in parentheses. Student’s paired t test was used to evaluate the statistical significance of the change in slope conductance; *P < 0·05; **P < 0·01. Figure 6. The effects of metabolic inhibition on the current—voltage relation Typical steady-state current—voltage relations under control conditions (1) and 3 min after application of 250 ìÒ DNP plus 10 mÒ deoxyglucose (0). The cross-over of the two curves was near the calculated K¤ equilibrium potential (arrow). J. Physiol. 525.2 KATP channels in capillaries expressed in capillary endothelial cells. At present we do not know whether Kir6.1 or Kir6.2, or both, are involved in the formation of endothelial KATP channels. Furthermore, we found that only one â_subunit (SUR2B) was expressed in capillary endothelium. The moderate effects of 300 ìÒ diazoxide found in our experiments are consistent with KATP channels containing SUR2B or SUR1, but not SUR2A (Isomoto & Kurachi, 1997; Yokoshiki et al. 1998, 1999). When heterologously expressed in human embryonic kidney cells, the SUR2BÏKir6.1 channels form nucleosidediphosphate-sensitive K¤ channels (KNDP channels; smooth-muscle type of KATP channels) with a conductance of 33 pS in symmetrical K¤ solution (Yamada et al. 1997; Fujita & Kurachi, 2000). In our experiments, the median of the slope conductance measured at −55 mV in the presence of 1 ìÒ rilmakalim was 900 pS. Assuming a very high open-state probability in the presence of a maximally effective concentration of rilmakalim, the average number of (possibly smooth-muscle type, NDPsensitive) KATP channels per endothelial cell is calculated to be about 27 (i.e. 900 pSÏ33 pS). Taking into account the possible experimental errors, we estimate the number of KATP channels per cell to be in the range 20—40. So far, endothelial KATP channels have been found only in primary culture (Janigro et al. 1993) or in freshly isolated endothelial cells (Katnik & Adams, 1995, 1997). It may well be that during longer term culture the expression of KATP channels ceases (Aguilar-Bryan et al. 1992). The results reported here confirm and extend previous findings obtained in coronary capillaries with a potential-sensitive dye (Langheinrich & Daut, 1997). However, the surprising effects of low concentrations of diazoxide on membrane potential, which have been consistently found in the fluorometric measurements, were not observed in the patchclamp experiments. In the present series of experiments, application of 300 nÒ diazoxide had no measurable effect. One possible explanation is that low concentrations of diazoxide have additional effects on intracellular organelles, for example, mitochondria, which are picked up in the fluorometric measurements but are not observed in the patch-clamp recordings. The possible functions of endothelial KATP channels We have previously shown that activation of KATP channels in coronary capillaries by K¤ channel openers elicits a rise in free intracellular Ca¥ and, in some cases, Ca¥ oscillations (Langheinrich et al. 1998). Ca¥ entry into endothelial cells is probably mediated by a passive pathway and is favoured by hyperpolarization (Cannell & Sage, 1989; Laskey et al. 1992). At present it is not clear what fraction of endothelial KATP channels is open under physiological or under pathophysiological conditions and how their open-state probability is regulated via endogenous vasoactive substances and intracellular second messengers (see Introduction). Various vasoactive substances that elicit an increase in free intracellular 315 Ca¥ cause an increase in the hydraulic conductivity and in the permeability of the vascular wall to macromolecules. These effects are mediated by the NOÏcGMP pathway downstream of the Ca¥ influx and can be prevented by blockade of NO synthase (Michel & Curry, 1999). A similar sequence of events may take place during hypoxia in the microvasculature. The changes in intracellular ATP and ADP lead to the opening of KATP channels and a subsequent hyperpolarization (Fig. 6). The resulting increase in free intracellular Ca¥ may give rise to a change in the barrier function of the vascular wall, mediated by the NOÏcGMP cascade. 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Hoffmann for excellent technical and secretarial help. This work was supported by the Deutsche Forschungsgemeinschaft (Da 177Ï7-2), the ‘Ernst und Berta Grimmke Stiftung’, the ‘Karl und Lore Klein Stiftung’ and the ‘P.E. Kempkes Stiftung’. Corresponding author J. Daut: Institut fur Normale und Pathologische Physiologie, Universitat Marburg, Deutschhausstrasse 2, D_35037 Marburg, Germany. Email: [email protected] 317