Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557
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ABSTRACT |
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Ca+/calmodulin-dependent
protein kinase II (CaM kinase II) is regulated by calcium oscillations,
autophosphorylation, and its subunit composition. All four subunit
isoforms were detected in gastric fundus and proximal colon smooth
muscles by RT-PCR, but only the and
isoforms are expressed in
myocytes. Relative
and
message levels were quantitated by
real-time PCR. CaM kinase II protein and
Ca2+/calmodulin-stimulated (total) activity levels are
higher in proximal colon smooth muscle lysates than in fundus lysates,
but Ca2+/calmodulin-independent (autonomous) activity is
higher in fundus lysates. CaM kinase II in fundus lysates is relatively
unresponsive to Ca2+/calmodulin. Alkaline phosphatase
decreased CaM kinase II autonomous activity in fundus lysates and
restored its responsiveness to Ca2+/calmodulin.
Acetylcholine (ACh) increased autonomous CaM kinase II activity in
fundus and proximal colon smooth muscles in a time- and dose-dependent
manner. KN-93 enhanced ACh-induced fundus contractions but inhibited
proximal colon contractions. The different properties of CaM kinase II
from fundus and proximal colon smooth muscles suggest differential
regulation of its autophosphorylation and activity in tonic and phasic
gastrointestinal smooth muscles.
calmodulin; protein kinase; phosphorylation; tonic smooth muscle; phasic smooth muscle
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INTRODUCTION |
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CA2+/calmodulin-dependent
protein kinase ii (CaM kinase II) mediates many cellular
responses to elevated Ca2+ in a wide variety of cells and
tissues (33). CaM kinase II is involved in the regulation
of ion channels, cytoskeletal dynamics, gene transcription,
neurotransmitter synthesis, insulin secretion, and cell division
(5a). In vascular smooth muscle tissues and cultured cells, CaM
kinase II is implicated in the modulation of myosin light chain kinase
sensitivity to Ca2+ and in regulation of cell migration,
and it is involved in the control of Ca2+ channels,
sarcoplasmic reticulum Ca2+-ATPase activity, and MAP kinase
activation (2, 3, 10, 24, 37). CaM kinase II is activated
by physiological Ca2+ elevations in vascular smooth muscle
cells, and its inhibition decreases contraction and force maintenance
by inhibiting MAP kinase activation and myosin light chain (LC20)
phosphorylation (2, 18, 26). In cardiac muscle
phosphorylation of phospholamban Thr17 by CaM kinase II in
response to -adrenergic stimulation activates the sarcoplasmic
reticulum Ca2+-ATPase, resulting in faster relaxation
(31). In addition, the membrane potential and excitability
of proximal colon smooth muscle cells are modulated by CaM kinase II
regulation of delayed rectifier K+ currents and
Ca2+-activated K+ channels (19,
20). These findings indicate that a number of proteins are
substrates for CaM kinase II in smooth muscle and suggest that CaM
kinase II can function at several points to enhance or attenuate the
contractile response.
CaM kinase II holoenzymes are multimers composed of 6-12 kinase
subunits arranged as a stacked pair of hexameric rings
(33). The central core of the holoenzyme contains the
COOH-terminal association domains of each subunit, with the variable
domain and NH2-terminal regulatory and catalytic domains
extending outward (33). Four genes encode the kinase
subunit isoforms (,
,
,
), and alternative splicing within
the variable domain generates additional diversity (39).
The
- and
-isoforms have narrow distributions, being restricted
to neuronal and endocrine tissues, whereas the
- and
-isoforms
are ubiquitously expressed within neuronal and nonneuronal tissues
(8). The functional significance of many of the variable
domains to the enzymatic activity of CaM kinase II is not clear.
Variable domain region I regulates the cytoplasmic distribution of the
-isoform and increases its affinity for Ca2+/calmodulin
(CaM) but has no effect on the affinity of the
A isoform
for Ca2+/CaM (21, 40). Variable domain region
III is responsible for the nuclear localization of
B CaM
kinase II (25, 40).
Ca2+/CaM binding to the holoenzyme kinase subunits results
in the rapid phosphorylation of Thr286 (numbering based on
the -isoform) on adjacent activated subunits (5a). Two consequences
of Thr286 autophosphorylation are that 1) the
rate of Ca2+/CaM dissociation in response to
Ca2+ removal is decreased by several orders of magnitude,
and 2) the kinase maintains activity toward its substrates
in the absence of Ca2+/CaM (independent or autonomous
activity) (5a). Thus the phosphorylation of target substrates by CaM
kinase II in response to transient increases in cytosolic
Ca2+ levels is sustained after the Ca2+ levels
decrease. The Ca2+-independent activity of CaM kinase II
plays an important role in the physiological response of cells to
transient Ca2+ increases (33). Additional
autophosphorylation in the CaM-binding domain at Thr305/306
following Thr286 autophosphorylation prevents subsequent
CaM binding and lowers total CaM kinase II activity levels (13,
14). Thr305/306 autophosphorylation also promotes
CaM kinase II disassociation from postsynaptic sites, providing a
mechanism for regulating the subcellular distribution of autonomous CaM
kinase II (30). The combined effects of these dual
autophosphorylations result in complex activation of the holoenzyme in
response to Ca2+ oscillations (7, 12, 13). The
level of autonomous activity of CaM kinase II in a tissue lysate is an
indication of prior activation of the enzyme because Thr286
autophosphorylation can only occur after Ca2+/CaM binding
to the holoenzyme. However, because Thr305/306
autophosphorylation regulates the number of kinase subunits that can
bind Ca2+/CaM, the extent of Thr286
autophosphorylation is also regulated by Thr305/306
autophosphorylation (12-14). Thus identical CaM
kinase II holoenzymes with different levels of Thr286 and
Thr305/306 autophosphorylation will have different kinetics
of CaM association and dissociation and of activation
(12-14). However, the subunit composition of the CaM
kinase II holoenzyme also regulates the kinetics of activation,
autophosphorylation, and CaM dissociation in response to
Ca2+ oscillations (5a, 7, 26). The holoenzyme structure of
CaM kinase II and the effects of Thr286 autophosphorylation
on enzyme activation led to the hypothesis that CaM kinase II can be
activated to different levels in response to different Ca2+
oscillation frequencies (12). The in vitro study of
immobilized CaM kinase II by De Koninck and Schulman provides strong
evidence supporting this hypothesis (7).
The proximal colon and gastric fundus are representative phasic and tonic gastrointestinal smooth muscles, respectively, that are characterized by different Ca2+ signaling patterns and sensitivities to excitation-contraction coupling (5, 34). Because CaM kinase II activity levels are modulated by Ca2+ oscillations, we are investigating the characteristics of CaM kinase II expressed in fundus and proximal colon smooth muscle tissues to test the hypothesis that tissues with different Ca2+ signaling patterns express CaM kinase II holoenzymes with different activation properties. Because the subunit composition influences activation, we investigated the CaM kinase II isoforms expressed in fundus and proximal colon smooth muscle tissues by Western blot and real-time PCR analysis. We investigated the regulation of CaM kinase II activation by Thr286 and Thr305/306 autophosphorylation by measuring the total and autonomous CaM kinase II activity levels and comparing the kinetics of generation of CaM kinase II autonomous activity in each smooth muscle tissue. Generation of autonomous CaM kinase II activity in fundus lysates required prior alkaline phosphatase treatment. Total CaM kinase II activity levels in fundus are also increased following alkaline phosphatase treatment. Our findings indicate that gastric fundus and proximal colon are characterized by CaM kinase II holoenzymes having distinct enzymatic characteristics and may be differentially regulated by Thr286 and Thr305/306 autophosphorylation. Incubation of fundus and proximal colon smooth muscle tissues with acetylcholine (ACh) increased autonomous CaM kinase II activities. The CaM kinase II inhibitor KN-93 enhanced the generation of tone in fundus and inhibited phasic contractions in proximal colon smooth muscle tissues in response to ACh stimulation. Together, these results indicate that CaM kinase II is activated by contractile stimuli and modulates contractile force in fundus and proximal colon smooth muscle tissues in vivo.
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MATERIALS AND METHODS |
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Materials.
Alkaline phosphatase and EDTA-free protease inhibitor tablets were
purchased from Roche (Indianapolis, IN). CaM was obtained from Sigma
(St. Louis, MO). [-32P]ATP (6,000 Ci/mmol) was
purchased from ICN (Costa Mesa, CA). Autocamtide-2 was obtained through
United Biomedical Research (Seattle, WA). Taq DNA
polymerase, dNTPs, and SuperScript II reverse transcriptase were
purchased from Life Technologies (Gaithersburg, MD). Primers were
synthesized by Life Technologies. Goat anti-CaM kinase II
,
,
, and
antibodies were purchased from Santa Cruz Biotechnology
(Santa Cruz, CA). Rabbit anti-PO4-Thr286
antibody (anti-active CaM kinase II) was purchased from Promega (Madison WI). This antibody recognizes the sequence surrounding the autophosphorylated Thr286 or Thr287 in
CaM kinase II
- and
-subunits
[MHRQET(PO4)VDCLKKFN]. Alkaline phosphatase-conjugated
rabbit anti-goat IgG antibodies were obtained from Chemicon (Temecula,
CA). L-Phenylalanine and ACh were from Sigma. KN-93 and
KN-92 were purchased from Biomol (Plymouth Meeting, PA) and Calbiochem
(San Diego, Ca), respectively. CD-1 mice were purchased from Charles
River (Cambridge, MA).
CaM kinase II activity assays.
Fundus and proximal colon smooth muscle tissues were prepared from the
stomachs and colons removed from adult CD-1 mice. Briefly, mice were
anesthetized with CHCl3, followed by cervical dislocation and removal of tissues as approved by the Institutional Animal Care and
Use Committee. The fundus and proximal colon tissues were pinned out in
a Sylgard-lined dish and washed with Ca2+-free Hanks'
buffer (125 mM NaCl, 5.36 mM KCl, 15.5 mM NaOH, 0.336 mM
Na2HPO4, 0.44 mM
KH2PO4, 10 mM glucose, 2.9 mM sucrose, and 11 mM HEPES, pH 7.2). The mucosa and submucosa layers were removed with
fine-tipped forceps. Fundus and proximal colon smooth muscle tissues
were obtained as described above and pinned out in small dishes
containing Krebs buffer (120 mM NaCl, 6 mM KCl, 15 mM
NaHCO3, 12 mM glucose, 3 mM MgCl2, 1.5 mM
NaH2PO4, and 3.5 mM CaCl2, pH 7.2).
For determining the effect of ACh on CaM kinase II activity, the
tissues were equilibrated in Krebs buffer for 1 h at 37°C and
then incubated at 37°C for various times in the absence or presence
of 10 µM ACh or for 30 min with various ACh concentrations. KN-93 was
added to the equilibrated tissues 20 min before the addition of ACh.
After treatment, the tissues were collected, frozen in liquid nitrogen,
and stored at 80°C until used for the CaM kinase II assays. When
needed for assays, frozen tissues were homogenized at 4°C with a
glass tissue grinder in lysis buffer (50 mM MOPS, 2% Nonidet P-40, 100 mM Na4P2O7, 100 mM NaF, 250 mM
NaCl, 3 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and a protease inhibitor tablet). Homogenates were
centrifuged at 16,000 g for 15 min at 4°C. The supernatant
was aliquoted and stored at
80°C until the kinase assay was
performed. Kinase assays were done at least three times in triplicate
from each tissue from three animals. Protein concentrations were
determined by using the Bradford assay with bovine gamma globulin as standard.
Dephosphorylation of fundus and proximal colon smooth muscle tissue lysates. Smooth muscle tissues were obtained and homogenized as described in CaM kinase II activity assays, with the exception that phosphatase inhibitors (Na4P2O7 and NaF) were omitted from the homogenization buffer. Lysates were incubated with calf intestinal alkaline phosphatase at 37°C for 40 min by using a range of 0.2-1.0 unit of enzyme per microgram of lysate. Phenylalanine was added to the samples (5 mM final concentration) to inhibit alkaline phosphatase (17). Assays for total and autonomous CaM kinase II activity were performed on control and dephosphorylated lysates as described above.
SDS-PAGE and Western blot analysis of CaM kinase II from proximal
colon and fundus smooth muscle tissues.
Fundus and proximal colon smooth muscle tissue lysates were obtained
from CD-1 mice, and protein concentrations were determined as described
in CaM kinase II activity assays. Tissue lysate
proteins were separated by SDS-PAGE (7.5%) and transferred to
nitrocellulose by Western blotting. The amount of lysate protein per
lane is indicated. The blots were incubated with primary and secondary antibodies, washed, and processed for image detection by using the
Western-Light chemiluminescence system from Tropix (Bedford, MA). The
CaM kinase II antibodies were used at 1:200 dilutions, and the alkaline
phosphatase-conjugated rabbit anti-goat IgG antibody was used at a
1:5,000 dilution. Protein bands were visualized with a charge-coupled
device camera-based detection system (Epi Chem II; UVP Laboratory
Products). The collected images were opened in Adobe Photoshop and
inverted for analysis. Densitometry was carried out by using Un-Scan-It
software from Silk Scientific. 316
CaM kinase II was
baculovirus-expressed and purified by CaM-Sepharose chromatography as
described previously (23). Autophosphorylation and
alkaline phosphatase treatment of purified
316
CaM kinase II was
carried out as described above.
RT-PCR of ,
,
, and
CaM kinase II isoforms from
proximal colon and fundus smooth muscle tissues.
Fundus and proximal colon smooth muscle tissues were obtained from CD-1
mice, and smooth muscle cells from each tissue were enzymatically
dispersed and collected as described (9). Total RNA was
purified by using the Epicenter MasterPure RNA purification kit.
First-strand cDNA was synthesized from 4.0 µg of total RNA by using
random primers and SuperScript II Moloney murine leukemia virus RNase
H
reverse transcriptase (Life Technologies). PCR was performed on 2 µl of cDNA with a final concentration of 1.5 mM MgCl2,
0.2 mM dNTPs, 0.5 µM primers, and 0.025 unit of Taq DNA
polymerase (Life Technologies). First-strand cDNA preparations were
monitored for the absence of genomic DNA by using intron-flanking
-actin primers (9). To identify splice variants, we
used primers designed to flank the variable region of each CaM kinase
II isoform. The
and
forward primers were designed at
nucleotides encoding for amino acids WDTVTPE
(5'-GGGACACAGTGACACCTGAA-3' and 5'-GGACACAGTCACTCCTGAA-3', respectively), the
reverse primer was complementary to bases encoding for amino acids EALGNLV (5'-CACTAAGTTGCCCAATGCTTC-3'), and the
reverse primer was complimentary to bases encoding for amino acids LGNLVE (5'-CTCCACGAGGTTACCAACC-3'). The forward
primer
corresponds to the nucleotides encoding amino acids LLASKL (5'-GTTGCTGGCTTCGAAGCTC-3'), and the
reverse primer is
complementary to the nucleotide sequence encoding amino acids GLDFHR
(5'-ATCGATGAAAGTCCAGCCC-3'). The forward
primer corresponds to the
nucleotides encoding amino acids EVLRKE (5'-CGAGGTCCTTCGGAAGGAGG-3'),
and the
reverse primer is complementary to the nucleotide sequence
encoding amino acids EALGNL (5'-GACCAGGTTGCCCAGAGCTT-3'). The PCR
profile for the
primers was as follows: 30 s of denaturation
at 94°C, 30 s of annealing at 63°C, and 1 min of extension at
72°C for 40 cycles, with a final 5-min extension at 72°C. The PCR
profile for the
,
, and
primers was the same as that for the
primers, except for annealing at 55°C, 61°C, and 63°C,
respectively. The PCR products were separated in 4.0% agarose gels and
visualized with GelStar stain (BioWhittaker Molecular Applications;
Rockland, CA.). The fragments were excised from the gel, isolated with
Micropure Separators (Amicon), and cloned with the Topo II TA cloning
kit (Invitrogen). Putative positive clones were identified by
diagnostic PCR. The plasmids were subsequently purified by using the
Quantum Prep plasmid miniprep kit (Bio-Rad), and the cloned PCR
fragments were sequenced using an ABI dye terminator cycle sequencer
(Applied Biosystems, Foster City, CA.).
Real-time PCR analysis of ,
,
, and
CaM kinase II
isoforms from proximal colon and fundus smooth muscle tissues.
Total RNA was purified as described above, and the pan
,
,
,
or
primers were used in the real-time PCR reactions. Relative quantitation of the
,
,
, or
CaM kinase II isoforms was
performed by using SYBR green detection real-time PCR. cDNA from CD-1
mouse brain cerebellum was initially diluted 1:20 and then serially diluted 1:2 for six dilutions, with 2 µl subsequently used as template for the standard curves of the CaM kinase II isoforms and the
internal standard, GAPDH. Proximal colon and fundus cDNA samples were
diluted 1:20 and 1:40, with 2 µl used as template in triplicate
reactions for the CaM kinase II isoforms and GAPDH. The SYBR green
master mix (Applied Biosystems) and the gene-specific pan primers at
final concentrations of 0.3 µM were used for amplification and
detection of product on an ABI Prism 7700 sequence detection system
(Applied Biosystems). The relative quantitation was then determined by
the standard curve method (ABI, User Bulletin no. 2).
Mechanical responses of fundus and proximal colon smooth muscle tissues. A standard organ bath technique was employed to measure changes in isometric force provided by fundus muscle strips. The mucosa was removed from the gastric fundus by sharp dissection, and strips of muscle (~6 × 3 mm) were isolated. One end of the tissue was attached to a fixed mount, and the opposite end to a Fort 10 isometric strain gauge (WPI, Sarasota, FL). The muscles were immersed in organ baths maintained at 37 ± 0.5°C with oxygenated Krebs solution (KRB). A resting force of 400 mg was applied to set the muscles at optimum length (data not shown). This was followed by an equilibration period of 1 h, during which time the bath was continuously perfused with oxygenated KRB. Mechanical responses were recorded on a personal computer running Acqknowledge 3.2.6 (BIOPAC Systems, Santa Barbara, CA).
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RESULTS |
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Identification of CaM kinase II isoforms in fundus and proximal
colon smooth muscle tissues.
Isoform-specific primer pairs that anneal to sequences common to each
reported isoform splice variant were used in RT-PCR analysis to first
determine which mRNAs are present for ,
,
, and
CaM kinase
II in murine proximal colon and fundus smooth muscle tissues. As shown
in Fig. 1A, PCR products were
obtained from the
,
,
, and
CaM kinase II primer pairs.
Sequencing confirmed that the PCR products obtained with each CaM
kinase II isoform-specific primer pair corresponded to
,
,
,
and
CaM kinase II, indicating that mRNAs for all four CaM kinase II isoforms are present in murine proximal colon and fundus smooth muscle
tissues. It is well established that the expression of
and
CaM
kinase II is restricted to neuronal and neuroendocrine cells and that
and
CaM kinase II are expressed in cardiac, skeletal, and
vascular smooth muscle cells (5a, 29, 32, 38). To determine which CaM
kinase II isoforms are expressed in gastrointestinal smooth muscle
cells, cDNA from isolated fundus and proximal colon smooth muscle cells
was analyzed by RT-PCR. With the use of cDNA from collected fundus
smooth muscle cells, no PCR products were obtained with the
and
CaM kinase II primers (Fig. 1C). In contrast, PCR
products were obtained with the
and
CaM kinase II primers.
These findings indicate that murine fundus and proximal colon smooth
muscle cells express
and
CaM kinase II. These findings also
suggest that the
and
CaM kinase II detected in the fundus and
proximal colon smooth muscle tissue samples are most likely expressed
in enteric neurons.
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Relative expression of CaM kinase II isoforms in fundus and
proximal colon smooth muscle tissues.
Real-time PCR analysis was used to quantitate the relative message
levels of the ,
,
, and
CaM kinase II expressed in murine
fundus and proximal colon smooth muscle tissues. The PCR products were
amplified by the appropriate isoform-specific pan primers, and amounts
produced were measured relative to GAPDH. As shown in Fig.
2, in fundus and proximal colon smooth
muscle tissues,
CaM kinase II message levels are more abundant than
CaM kinase II message levels, relative to GAPDH message levels. The
:
ratio in fundus is ~1.66:1, whereas the
:
ratio in
proximal colon smooth muscle tissues is ~1.3:1. Relative to GAPDH and
to
and
CaM kinase II, the message levels of the
- and
-isoforms are present at extremely low levels in fundus and proximal
colon smooth muscle tissues. These findings are consistent with the RT-PCR results (Fig. 1C) showing that message for the
-
and
-isoforms was not amplified from isolated smooth muscle cells.
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Detection of CaM kinase II isoforms in murine fundus and proximal
colon smooth muscle tissues.
The results of the SDS-PAGE and Western blot analysis of gastric fundus
and proximal colon smooth muscle tissue lysates using CaM kinase II
isoform-specific antibodies are shown in Fig.
3. Murine brain lysates were used as a
positive control for and
CaM kinase II expression (5a, 26). In
agreement with previous reports, a strong signal at ~50 kDa was
generated from brain lysate by using the anti-
CaM kinase II
antibody (5a, 26). Similarly, a protein band at ~60 kDa was observed
from brain lysates by using the anti-
CaM kinase II antibody. The
CaM kinase II staining is less intense than the
CaM kinase II
staining from brain lysates. These results are consistent with previous
findings that
CaM kinase II is the predominant isoform expressed in
neuronal tissues (5a, 26). The anti-
CaM kinase II antibody
generated a modest signal at 50 kDa from proximal colon lysates and an
almost undetectable signal from fundus lysates. Protein staining of
fundus and proximal colon smooth muscle tissue lysates using the
anti-
CaM kinase II antibody was not detected. These results
indicate that proximal colon smooth muscle tissue is characterized by a
higher level of
CaM kinase II expression than fundus and suggest
that
CaM kinase II is not expressed or is expressed at levels below
the limits of detection in both smooth muscle tissues (see below). It
should be noted here that the amount of protein from fundus and
proximal colon lysates in each lane is twice the amount of the protein
in the lanes with brain lysates. These results indicate that the
and
CaM kinase II expression levels in fundus and proximal colon
smooth muscle tissues are lower than the expression levels found in
brain.
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Identification of CaM kinase II isoform splice variants in fundus
and proximal colon smooth muscle tissues.
RT-PCR analysis was carried out by using isoform-specific primers
flanking the variable region of CaM kinase II to identify the ,
,
, and
splice variants expressed in murine proximal colon and
fundus smooth muscle tissues. After agarose gel electrophoresis, the
PCR products were excised from the gels and cloned by using Topo
cloning vectors. As shown in Fig.
4A, four similarly sized PCR
products ranging from ~550 to 750 bp and 450 to 700 bp were obtained
from fundus and proximal colon smooth muscle tissues with the
- and
-specific primers, respectively. Sequencing the PCR products
obtained with the
-isoform-specific primers identified the 550-, 650-, 700-, and 750-bp PCR products as
C,
J,
B, and
I, respectively.
In contrast, although four PCR products are visible in the gel,
following cloning and sequencing, all five
CaM kinase II splice
variants were identified. Sequencing identified the 450-, 550-, 600-, and 700-bp PCR products from the
-isoform-specific primers as
C,
B/
E,
D,
and
A, respectively. The predicted sizes of the
B and
E amplicons differ from each other
by only nine base pairs; thus these two amplicons are unlikely to be
separated by the agarose gel electrophoresis. In addition, by using the variable domain-flanking primers specific for
CaM kinase II, we
amplified two PCR products of ~285 and 318 bp from fundus and proximal colon smooth muscle tissues and determined them to be
and
33 CaM kinase, respectively, by sequence analysis. Five PCR products
were obtained by using the variable domain-flanking primers specific
for
CaM kinase II. After cloning and sequencing, on the basis of
the predicted sizes of the splice variant amplicons, the 377-, 404-, 449-, and 635-bp products correspond to
'e,
e,
', and
CaM kinase II, respectively. Sequence
analysis indicates that the 318-bp amplicon corresponds to
33,
indicating that the
-isoform-specific primers also annealed to the
complementary sequences of
33 CaM kinase II. The nomenclature of the
,
,
, and
splice variants is from Tombes and Krystal
(39).
I and
J were
previously identified in rabbit liver as novel splice variants and were
named
H and
I, respectively
(36). Our results are the first demonstration of the
expression of these two
splice variants outside the liver,
indicating that they have a wider tissue distribution. Figure
4B shows a chart of the variable region domain structure of
these
and
splice variants and their corresponding predicted
amino acid sequences (39).
|
Total and autonomous CaM kinase II activity in fundus and proximal
colon smooth muscle lysates.
Because the subunit composition and levels of autophosphorylation
influence the kinase activity of the CaM kinase II holoenzyme, we
compared total and autonomous CaM kinase II activity from murine fundus
and proximal colon smooth muscle tissue lysates using the peptide
substrate autocamtide-2. Addition of the specific CaM kinase II
inhibitor KN-93 (1 µM) to the tissue lysates inhibited 90% of the
total kinase activity toward autocamtide-2 (data not shown), indicating
that, similar to previous reports, the autocamtide-2 kinase activity
measured in the tissue lysates is due to CaM kinase II (26,
35). Total CaM kinase II activity in fundus smooth muscle
lysates is threefold lower than the activity in proximal colon smooth
muscle tissue lysates (1.09 ± 0.19 vs. 3.65 ± 0.74 pmol · min1 · µg
1,
respectively). In contrast, autonomous CaM kinase II activity in fundus
is 3.5-fold higher than the autonomous activity in proximal colon
smooth muscle tissue (0.32 ± 0.07 vs. 0.09 ± 0.03 pmol · min
1 · µg
1,
respectively). Thus, as a percentage of total CaM kinase II activity,
autonomous CaM kinase II activity is high in fundus smooth muscle
tissues (28.9 ± 3.35%) but low in proximal colon smooth muscle
tissues (3.6 ± 0.6%). These findings indicate that fundus smooth
muscle tissue contains higher levels of autonomous CaM kinase II
activity than proximal colon smooth muscle tissue.
|
|
Activation of fundus smooth muscle CaM kinase II by alkaline
phosphatase.
A simplified diagram of the current model of CaM kinase II regulation
by autophosphorylation is diagrammed in Fig.
7. Ca2+/CaM can cause little
or no additional Thr286 autophosphorylation and autonomous
activity as the number of Thr286-autophosphorylated kinase
subunits of a holoenzyme increases (Fig. 7, II and
III). This model and our results showing that autonomous CaM
kinase II activity in fundus smooth muscle lysates is 30% of the total
CaM kinase II activity and is largely resistant to generation of
additional autonomous activity suggest that fundus smooth muscle CaM
kinase II may be almost fully Thr286 phosphorylated (Fig.
7, III). Evidence in support of this conclusion is provided
by the results of the experiments comparing autonomous CaM kinase II
activity before and after alkaline phosphatase treatment. We first
confirmed that alkaline phosphatase dephosphorylates Thr286-autophosphorylated CaM kinase II using
autophosphorylated 316
CaM kinase II. As shown in Fig.
8, lane 1, the
anti-PO4-Thr286 antibody generated a strong
signal toward autophosphorylated
316
CaM kinase II. However, no
signal was detected following incubation of autophosphorylated
316
CaM kinase II with alkaline phosphatase (Fig. 8, lane
2). These results demonstrate that alkaline phosphatase
dephosphorylates Thr286. Next, we examined the ability of
alkaline phosphatase to dephosphorylate CaM kinase II in fundus and
proximal colon smooth muscle lysates and the ability of CaM kinase II
to undergo Thr286 autophosphorylation following alkaline
phosphatase treatment of fundus and proximal colon smooth muscle
lysates. Lysates were incubated with alkaline phosphatase, followed by
phenylalanine (5 mM final concentration) to inhibit alkaline
phosphatase, as previously reported (17). The lysates were
then incubated CaM, CaCl2, and ATP to allow
Thr286 autophosphorylation to occur. No signal was detected
from alkaline phosphatase-treated fundus and proximal colon smooth
muscle lysates with the anti-PO4-Thr286
antibody (Fig. 8B, lanes 1 and 4).
However, strong signals were generated from the lysates that were
incubated alkaline phosphatase, followed by phenylalanine, and CaM,
CaCl2, and ATP (Fig. 8, lanes 2 and
3). These results demonstrate that alkaline phosphatase dephosphorylates Thr286 in fundus and proximal colon smooth
muscle tissue lysates. These results also demonstrate the ability of
CaM kinase II in fundus and proximal colon smooth muscle tissue lysates
to undergo Thr286 autophosphorylation following inhibition
of alkaline phosphatase by 5 mM phenylalanine.
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|
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Activation of CaM kinase II in fundus and proximal colon smooth
muscle tissues by ACh.
CaM kinase II is involved in the generation of contraction and
maintenance of force in vascular smooth muscle (18, 26). In addition, the in situ Ca2+ dependence for CaM kinase II
activation in cultured vascular smooth muscle cells falls within the
range of cytosolic Ca2+ concentration increases induced by
angiotensin II (1). However, our findings that CaM kinase
II from fundus smooth muscle lysates is unresponsive to
Ca2+/CaM activation compared with the enzyme from proximal
colon smooth muscle lysates suggest that CaM kinase II in fundus and
proximal colon smooth muscles may show different sensitivities to
physiological stimuli that increase cytosolic Ca2+ levels
in these tissues. Because ACh is the neurotransmitter primarily
responsible for Ca2+ mobilization and contraction of
gastrointestinal smooth muscle tissues, we assessed the ability of ACh
to activate CaM kinase II in fundus and proximal colon smooth muscle
tissues. Fundus and proximal colon smooth muscle tissues were cultured
in the absence or presence of 10 µM ACh, as described in
MATERIALS AND METHODS. CaM kinase II activation was
determined by measuring autonomous CaM kinase II activity in lysates
from control and ACh-treated tissues. As shown in Fig.
10, CaM kinase II autonomous activity
levels were elevated in a time- and dose-dependent manner in
ACh-treated fundus and proximal colon tissues. After a 45-min incubation of fundus smooth muscle tissues with 10 µM ACh, autonomous CaM kinase II activity increased from 31 to 54% of total CaM kinase II
activity. Similarly, incubation of proximal colon smooth muscle tissues
with 10 µM ACh increased autonomous CaM kinase II activity from 7 to
19% of total CaM kinase II activity (Fig. 10A). In
addition, autonomous CaM kinase II activity levels in both smooth
muscle tissues increased with increasing ACh concentrations and
plateaued between 5 and 10 µM ACh (Fig. 10B).
Preincubation of the smooth muscle tissues with KN-93 (1 µM)
prevented the ACh-induced increases in CaM kinase II autonomous
activities (data not shown). These findings indicate that contractile
stimuli activate CaM kinase II in fundus and proximal colon smooth
muscle tissues.
|
Effects of KN-93 on contractions of gastric fundus and proximal
colon smooth muscles.
Because ACh activated CaM kinase II, the contribution of CaM kinase II
to the contractile activity of the circular muscle layers of gastric
fundus and proximal colon was determined. ACh (10 µM) caused the
contractile tone of fundus smooth muscle to increase by 4.11 ± 0.8 mN (Fig. 11A). This
increase in tone was reversible upon washout of ACh. Perfusion of
muscle strips with KN-93 (5 µM) did not produce any resolvable change
in basal tension. After a 30-min perfusion period with KN-93, the
increase in contractile force produced by ACh increased to 7.9 ± 1.3 mN (Fig. 11B). Thus preincubation with KN-93 caused an
~90% increase in the amplitude of contractile force elicited by ACh.
Perfusion of the proximal colon with ACh (10 µM) produced an increase
in the tone of the circular muscle layer by 11.1 ± 1.7 mN and
also increased the amplitudes of the phasic contractions (Fig.
11C). In contrast to the circular muscle layer of the
fundus, incubation of proximal colon circular muscle with 1 µM KN-93
decreased spontaneous mechanical activity and reduced the tone produced
by ACh by 2.9 ± 0.4 mN. KN-93 also reduced the steady-state
amplitudes of ACh-induced phasic contractions to (Fig. 11D).
The inactive KN-93 analog KN-92 (5 µM) had no effect on basal and
ACh-induced contractions of fundus and proximal colon smooth muscles
(data not shown). These findings indicate differential roles for CaM
kinase II in regulating contractile activity of gastric fundus and
proximal colon smooth muscle tissues.
|
![]() |
DISCUSSION |
---|
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---|
The holoenzyme structure and subunit composition of CaM kinase II modulate its activation in response to Ca2+ oscillations (7). Although each subunit is independently activated by Ca2+/CaM, the kinase subunits regulate each other by intersubunit autophosphorylation reactions on Thr286 and Thr305/306 (28, 33). The number of Thr286-autophosphorylated subunits determines the sensitivity of the CaM kinase II holoenzyme to Ca2+/CaM stimulation, and the level and duration of autonomous activity (7). Different frequencies and amplitudes of Ca2+ oscillations give rise to different levels of Thr286 autophosphorylation and to CaM kinase II holoenzymes with different levels of autonomous activity and sensitivities to Ca2+/CaM stimulation (7). These findings suggest that tissues and cells having different Ca2+ oscillation patterns may express CaM kinase II holoenzymes characterized by different subunit compositions and levels of autophosphorylation. To test this hypothesis, we have initiated studies to characterize CaM kinase II in gastric fundus and proximal colon smooth muscle tissues. The fundus and proximal colon are tonic and phasic gastrointestinal smooth muscle tissues, respectively, with distinct Ca2+ signaling characteristics (5, 34). Tonic smooth muscles generally have higher resting Ca2+ levels compared with phasic smooth muscles (30). These differences in Ca2+ signaling underlie the cyclic depolarizations and repolarizations that determine the phasic contractile activity of the colon and also the tonic activity of the gastric fundus due to neural and hormonal regulation (16, 34).
RT-PCR analysis demonstrated the presence of message for ,
,
,
and
CaM kinase II in fundus and proximal colon smooth muscle
tissues (Fig. 1). In contrast, in isolated smooth muscle cells, the
RT-PCR analysis shows only
and
CaM kinase II expression (Fig.
1C). These findings indicate that smooth muscle cells of the
digestive tract express
and
CaM kinase II. These findings also
suggest that
and
CaM kinase II are expressed in the enteric neurons, because it has been previously established that
and
expression is restricted to neurons (5a). A strong
CaM kinase II
signal was obtained from brain lysates, whereas weaker signals were
generated from fundus and proximal colon smooth muscle lysates. Although we detected
CaM kinase II in Western blots of murine brain
lysates (Fig. 3), no signal was obtained from fundus and proximal colon
smooth muscle tissue lysates. It is well established that CaM kinase II
message levels correlate with protein levels (4). Thus the
lack of a
CaM kinase II signal from fundus and proximal colon
smooth muscle lysates is most likely due to a level of expression that
is below the limits of detection by Western blot analysis. Indeed, the
real-time PCR results indicate that the expression of CaM kinase II
and
expression is >100-fold lower than
and
expression in
fundus and proximal colon smooth muscle tissues. In the brain, CaM
kinase II mRNA is abundant and protein expression levels approach 2%
of total (33). Similarly, immunohistochemical results
suggest that
and
CaM kinase II may be expressed at high levels
in enteric neurons (19). However, neuronal mRNA represents
a small minority of the total mRNA purified from the fundus and
proximal colon smooth muscle tissues, with the vast majority of mRNA
purified from the smooth muscle cells.
Several subtypes of each CaM kinase II isoform are generated by
alternative splicing (29, 34). Using RT-PCR analysis, we
found the same two , four
, five
, and four
CaM kinase II
isoform splice variants expressed in murine fundus and proximal colon
smooth muscle tissues:
B,
C,
I,
J,
A,
B,
C,
D,
E,
,
33,
,
',
e, and
'e. These results do not support the hypothesis that tissues having different Ca2+ oscillation patterns
express different CaM kinase II subunit isoforms. However, the
real-time PCR analysis indicates that both fundus and proximal colon
have slightly different
:
ratios. We found
:
ratios of
~1.66:1 and 1.3:1 in fundus and proximal colon smooth muscle tissues,
respectively. Although these differences are less than twofold, they
may have significant functional implications for the CaM kinase II
holoenzyme subunit compositions. For example, a heptameric CaM kinase
II holoenzyme with a 1.3:1
:
ratio indicates that four subunits
are
and three are
. In contrast, a 1.66:1
:
ratio suggests
an octameric CaM kinase II holoenzyme with five
and three
subunits. We are currently carrying out coimmunoprecipitation experiments to determine the presence of
and
CaM kinase II heteromeric holoenzymes in fundus and proximal colon smooth muscle tissues.
Consistent with the real-time PCR results and previous reports of CaM
kinase II expression in cardiac and vascular smooth muscle, Western
blot analysis indicates that and
CaM kinase II protein
expression are also prevalent in murine fundus and proximal colon
smooth muscle tissues. The apparent mass (~60 kDa) of the protein
bands stained with the anti-
CaM kinase II antibody suggest that
A is the predominant subtype expressed in murine fundus
and proximal colon smooth muscles. The single protein band detected
with the anti-
CaM kinase II antibody is probably due to the
subtypes detected by RT-PCR in murine fundus and proximal colon smooth
muscles (
B,
C,
I,
J), which have been found to range in mass from 59 to 63 kDa (34). The densitometric analysis of the
and
CaM kinase II protein bands from the Western blots indicate
that CaM kinase II protein expression is higher in proximal colon than
in fundus.
The pattern of and
CaM kinase II splice variant expression is
unique to fundus and proximal colon smooth muscle tissues. Astrocytes
express CaM kinase II isoform splice variants
A,
B, and
C (41). Aortic
myocytes express CaM kinase II
B,
C,
E,
G, and
C
(32). Biliary epithelial cells express only CaM kinase II
splice variants (22). CaM kinase II
B,
C,
I,
J,
C,
C6, and
C,
C6 with
alternative COOH termini are expressed in rabbit liver
(36). The identification of CaM kinase II
I
and
J in fundus and proximal colon smooth muscle tissues
is the first demonstration of the expression of these two CaM kinase II
splice variants outside the liver, suggesting that they have a
wider tissue distribution.
Although the same CaM kinase II ,
,
, and
splice variants
were identified in fundus and proximal colon smooth muscle tissue, we
observed distinct differences in CaM kinase II enzymatic activity from
these two tissues. Total CaM kinase II activity levels in proximal
colon smooth muscle tissue lysates were approximately threefold higher
than total CaM kinase II activity levels in fundus smooth muscle
tissue. However, fundus smooth muscle lysates are characterized by a
high level of autonomous CaM kinase II activity compared with proximal
colon smooth muscle lysates (23-32% vs. 4-10% of total CaM
kinase II activity). Both tissues were processed for kinase assays
identically, suggesting that the high levels of autonomous CaM kinase
II in fundus lysates are not due to proteolysis of CaM kinase II during
tissue preparation. To demonstrate that Thr286
autophosphorylation is responsible for the high level of autonomous CaM
kinase II activity, we treated fundus smooth muscle lysates with
alkaline phosphatase to dephosphorylate Thr286. As shown in
Fig. 8, autonomous CaM kinase II activity levels in fundus smooth
muscle tissue lysates decreased from ~23 to 7% of total CaM kinase
II activity. These results provide strong evidence that the high level
of autonomous CaM kinase II activity in fundus smooth muscle tissue
lysates is due to Thr286 autophosphorylation. The low level
of autonomous CaM kinase II activity in proximal colon smooth muscle
tissue lysates was also decreased slightly by alkaline phosphatase
(data not shown). The remaining kinase activity measured in the tissue
lysates following alkaline phosphatase treatment may be due to other
kinases or to a limited amount of CaM kinase II proteolyzed
endogenously or during tissue preparation.
In addition to differences in total and autonomous activity, CaM kinase II activity in fundus and proximal colon smooth muscle tissue lysates displayed differences in sensitivities to Ca2+/CaM activation. Ca2+/CaM stimulation of CaM kinase II in proximal colon smooth muscle tissue lysates increased autonomous CaM kinase II activity from 4 to 52% of total CaM kinase II activity. However, Ca2+/CaM stimulation only increased CaM kinase II autonomous activity to 37% from 29% in fundus smooth muscle lysates (Fig. 6). The results in Fig. 8 showing that alkaline phosphatase decreases autonomous CaM kinase II activity indicate that the high level of autonomous CaM kinase II activity in fundus smooth muscle tissue lysates is due to Thr286 autophosphorylation. Little or no additional autonomous activity can be generated by Ca2+/CaM activation if all (or most) of the kinase subunits of a CaM kinase II holoenzyme are already Thr286 autophosphorylated. Our findings that CaM kinase II in fundus smooth muscle lysates is resistant to Ca2+/CaM-induced generation of autonomous activity (Fig. 6) further suggest that fundus CaM kinase II is characterized by a high level of Thr286 autophosphorylation.
The lower levels of total CaM kinase II activity in fundus relative to
proximal colon smooth muscle tissues could be due to differences in CaM
kinase II protein levels between the two tissues, different holoenzyme
subunit compositions, or differences in levels of
Thr305/306 phosphorylation. CaM kinase II subunits that are
autophosphorylated on Thr305/306 cannot bind
Ca2+/CaM. Thus the total activity from a CaM kinase II
holoenzyme with some (or all) subunits Thr305/306
phosphorylated is lower than the total activity of a CaM kinase II
holoenzyme having none of its kinase subunits Thr305/306
phosphorylated (13, 14). To determine whether the lower
level of total CaM kinase II activity in fundus smooth muscle lysates is due to autophosphorylated Thr305/306, we incubated
fundus and proximal colon smooth muscle lysates with alkaline
phosphatase to dephosphorylate any
Thr305/306-phosphorylated CaM kinase II subunits before the
addition of Ca2+/CaM and the subsequent determination of
total CaM kinase II activity. Alkaline phosphatase treatment of fundus
lysates caused a fourfold increase in total CaM kinase II activity. In
contrast, a less than twofold increase in total CaM kinase II activity
was measured in alkaline phosphatase-treated proximal colon smooth
muscle lysates. The findings from the alkaline phosphatase studies
suggest that fundus smooth muscle CaM kinase II holoenzymes contain a
high level of phosphorylated Thr305/306, which lowers total
CaM kinase II activity. However, after alkaline phosphatase treatment,
the total CaM kinase II activity levels in fundus lysates were still
1.3-1.5 times lower than the total CaM kinase II activity levels
in proximal colon lysates. Furthermore, the densitometric analyses of
the Western blots suggest that the and
CaM kinase II protein
expression levels in fundus smooth muscle tissues are about 1.4-fold
less than the levels in proximal colon. Together, these findings
suggest that fundus smooth muscle tissue has lower levels of CaM
kinase II protein expression than proximal colon smooth muscle tissue.
The data do not support the conclusion that fundus smooth muscle CaM
kinase II is fully Thr305/306 phosphorylated. The total CaM
kinase II activity is higher than the autonomous activity, indicating
that Ca2+/CaM is binding to and activating the holoenzyme.
It should be noted that kinetic analyses of purified CaM kinase II have
been used to distinguish non-Thr305/306-phosphorylated from
Thr305/306-phosphorylated CaM kinase II (13).
These studies have shown that when Ca2+/CaM is bound to the
CaM kinase II holoenzyme, the Km for the autocamtide-2 peptide substrate is 0.3 µM. However, in the absence of
Ca2+/CaM, the Km of autonomous CaM
kinase II for autocamtide-2 is 3 µM (13). Because
Thr305/306-phosphorylated CaM kinase II cannot bind
Ca2+/CaM, its Km for autocamtide-3
is 3 µM in the presence of Ca2+/CaM (13).
However, we cannot use kinetic analyses to investigate Thr305/306 phosphorylation of fundus and proximal CaM
kinase II holoenzymes. In each tissue, the CaM kinase II holoenzymes
are not fully phosphorylated on Thr286 and
Thr305/306, unlike the in vitro studies (12,
13). In addition, the CaM kinase II protein levels in each
tissue are unknown, so it is not possible to assay and compare equal
amounts of CaM kinase II in kinetic assays.
ACh binding to m2 and m3 muscarinic receptors on gastrointestinal smooth muscle cells causes contraction by Ca2+ influx through L-type Ca2+ channels and inositol 1,4,5-trisphosphate-induced Ca2+ release (27). ACh treatment of fundus and proximal colon smooth muscle tissues activates CaM kinase II as indicated by our results showing increases in autonomous CaM kinase II activity levels. KN-93 inhibited the phasic contractile activity of proximal colon smooth muscles induced by ACh. In contrast, KN-93 enhanced the ACh-induced increase in tonic contractions of fundus smooth muscle. These findings suggest different functional roles for CaM kinase II in modulating the contractile activities of fundus and proximal colon smooth muscles. Our results with the CaM kinase II inhibitor KN-93 suggest that CaM kinase II activation inhibits fundus contractions but enhances proximal colon contractions. CaM kinase II sensitizes the contractile apparatus of vascular smooth muscle to Ca2+ via MAP kinase activation and LC20 phosphorylation (18, 26). In addition, KN-93 inhibited CaM kinase II activation and LC20 phosphorylation (18, 26). These findings and our results (Fig. 11) suggest that CaM kinase II activation and regulation of contraction is a common feature of smooth muscle contraction. Although ACh increased autonomous CaM kinase II activity in fundus smooth muscle tissues from 30 to 55% of total activity, our results indicate that CaM kinase II in fundus lysates is unresponsive to Ca2+/CaM-induced activation and increases in autonomous activity compared with CaM kinase II in proximal colon lysates. Additional studies are underway to elucidate the mechanism of CaM kinase II activation and its substrates during ACh-induced contractions in fundus and proximal colon smooth muscles.
In summary, we have demonstrated that transcripts for two and four
CaM kinase II isoforms are expressed in murine gastric fundus and
proximal colon smooth muscle tissues. In addition, we found a pattern
of expression of the
and
isoforms unique to these two smooth
muscle tissues. Expression of the
and
CaM kinase II isoforms
appears to be prevalent in fundus and proximal colon smooth muscle
tissues by Western blot analysis. ACh activated CaM kinase II in fundus
and proximal colon smooth muscle tissues. The CaM kinase II inhibitor
KN-93 enhanced ACh-induced contractions of fundus but inhibited
contractions of proximal colon smooth muscles induced by ACh. Although
both tissues express the same
,
,
, and
CaM kinase II
isoforms, the two tissues exhibited differences in CaM kinase II
expression levels and total and autonomous CaM kinase II activity
levels. Although total CaM kinase II activity levels are lower in
fundus than in proximal colon smooth muscle, the percentage of
autonomous CaM kinase II activity in fundus smooth muscle is higher.
Proximal colon smooth muscle CaM kinase II readily generated additional
autonomous activity. In contrast, gastric fundus smooth muscle CaM
kinase II was largely unresponsive to Ca2+/CaM activation
and required alkaline phosphatase treatment to generate additional
autonomous activity. Alkaline phosphatase treatment of fundus smooth
muscle lysates lowered autonomous and increased total CaM kinase II
activity levels. These results suggest that CaM kinase II holoenzymes
in fundus smooth muscle tissues are characterized by a high level of
Thr286 and Thr305/306 autophosphorylation. The
proximal colon and fundus smooth muscle tissues used in these studies
to measure total and autonomous CaM kinase II activities and identify
the expressed CaM kinase II isoforms contain the circular and
longitudinal smooth muscle layers and are predominantly composed of
myocytes. However, interstitial cells of Cajal and enteric ganglia are
also present interspersed among the myocytes (16). Further
investigations using in situ hybridization and immunohistochemical
analyses are necessary to determine the cellular distribution of the
CaM kinase II isoforms and autonomous CaM kinase II in gastric fundus
and proximal colon smooth muscle tissues. In addition, future studies
must investigate the basis for the differences in CaM kinase II
autophosphorylation at Thr286 and Thr305/306 in
gastric fundus and proximal colon smooth muscle tissues.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Sang Don Koh for the technique of dispersing and collecting smooth muscle cells and for helpful discussions. We thank Dr. Burton Horowitz and Dr. Sean Ward for critical reading of the manuscript. We also thank Dr. James Kenyon for critical reading of the manuscript and many insightful discussions.
![]() |
FOOTNOTES |
---|
* J. M. Lorenz and M. H. Riddervold contributed equally to this work.
This work was supported by National Institutes of Health Grants DK-57168 and NS-36318.
Address for reprint requests and other correspondence: B. A. Perrino, Dept. of Physiology and Cell Biology, Univ. of Nevada School of Medicine, Anderson Bldg./MS352, Reno, NV 89557 (E-mail: perrino{at}physio.unr.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
July 3, 2002;10.1152/ajpcell.00020.2002
Received 15 January 2002; accepted in final form 26 June 2002.
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