Download ATP-sensitive potassium channels in capillaries isolated from

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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, J‡urgen Daut
and Regina Preisig_M‡uller
Institut f‡ur Normale und Pathologische Physiologie, Universit‡at 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-M‡uller
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-M‡uller 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 Regierungspr‡asidium, 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-M‡uller 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-M‡uller
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-M‡uller 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-M‡uller et al. 1999c).
312
M. Mederos y Schnitzler, C. Derst, J. Daut and R. Preisig-M‡uller
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-M‡uller
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
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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. Furthermore, the increased synthesis of
NO is expected to modulate the contractility of
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al. 1993), mitochondrial energy metabolism (Kaasik et al.
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al. 1999). Thus, the activation of KATP channels in coronary
capillaries via metabolic inhibition andÏor vasoactive factors
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Acknowledgements
We thank B. Burk, A. Schubert, R. Graf, K. Schneider,
A. Hennighausen, R. Luzius, A. Mazzola and E. 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 f‡ur Normale und Pathologische Physiologie,
Universit‡at Marburg, Deutschhausstrasse 2, D_35037 Marburg,
Germany.
Email: [email protected]
317