Departments of 1 Physiology and 2 Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9040; and 3 Department of Biochemistry & Biophysics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6059
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ABSTRACT |
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Phosphatidylinositol
4,5-bisphosphate (PIP2) affects profoundly several cardiac
ion channels and transporters, and studies of
PIP2-sensitive currents in excised patches suggest that
PIP2 can be synthesized and broken down within 30 s.
To test when, and if, total phosphatidylinositol 4-phosphate (PIP) and
PIP2 levels actually change in intact heart, we used a new,
nonradioactive HPLC method to quantify anionic phospholipids. Total PIP
and PIP2 levels (10-30 µmol/kg wet weight) do not
change, or even increase, with activation of
Gq/phospholipase C (PLC)-dependent pathways by carbachol
(50 µM), phenylephrine (50 µM), and endothelin-1 (0.3 µM).
Adenosine (0.2 mM) and phorbol 12-myristate 13-acetate (1µM) both
cause 30% reduction of PIP2 in ventricles, suggesting that
diacylglycerol (DAG)-dependent mechanisms negatively regulate cardiac
PIP2. PIP2, but not PIP, increases reversibly
by 30% during electrical stimulation (2 Hz for 5 min) in guinea pig
left atria; the increase is blocked by nickel (2 mM). Both PIP and
PIP2 increase within 3 min in hypertonic solutions, roughly
in proportion to osmolarity, and similar effects occur in multiple cell
lines. Inhibitors of several volume-sensitive signaling mechanisms do not affect these responses, suggesting that PIP2 metabolism
might be sensitive to membrane tension, per se.
phosphatidylinositol 4,5-bisphosphate; phosphatidylinositol; diacylglycerol; phorbol ester; cardiac muscle; G protein-coupled receptors; phospholipase C; cell volume
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INTRODUCTION |
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PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE (PIP2) is the phospholipid precursor of three second messengers, D-myo-inositol 1,4,5-trisphosphate (IP3), diacylglycerol (DAG), and phosphatidylinositol 3,4,5-trisphosphate (PIP3) (66). At the same time, PIP2 serves other cellular functions. It anchors and modulates the function of numerous cell signaling proteins and cytoskeleton at the cell membrane (11, 17, 42, 65), including at least one transcription factor that is released by phospholipase C (PLC) activation (60). In addition, PIP2 metabolism is coupled to membrane trafficking, including some forms of exo- and endocytosis (7, 46). Finally, PIP2 modulates the function of phospholipases (14), receptor kinases (16, 52), and ion transporters and ion channels (25). Especially, the anchoring/recruitment functions and the modulatory functions of PIP2 beg the question as to how, and if, PIP2 might be used as a cell signal. For cardiac physiology, an answer to this question seems especially important at this time, because sarcolemmal mechanisms that affect both cardiac contraction (e.g., Na+/Ca2+ exchange) and contraction frequency [e.g., G protein-coupled inwardly rectifying K+ (GIRK) channels] are strongly PIP2 dependent (27).
The minimum biochemical mechanisms involved in cardiac myocyte
PIP2 metabolism (39) are summarized in Fig.
1. The dominant pathway of
PIP2 synthesis, as in other cells, is probably the sequential phosphorylation in the sarcolemma of phosphatidylinositol (PI) at the 4- and then the 5-positions of inositol (66).
As in other cells, PIP2 is hydrolyzed by PLCs to generate
IP3 and DAG, or it can be dephosphorylated to PIP and PI.
DAG can be phosphorylated to generate phosphatidic acid (PA) by DAG
kinases, which may be regulated by translocation to the surface
membrane (32, 45), and dephosphorylation of PA can
probably be an important source of DAG in heart (for example, see Ref.
8). On internal membranes, PA serves as the phospholipid
precursor of PI, and sarcoplasmic reticulum is a major site of PI
synthesis in heart (70). As in other cells, PI in cardiac
myocytes is probably transported to the surface membrane by transfer
proteins (PITP) (70) as well as by constitutive membrane
trafficking during the recycling of membranes to endosomes (not shown).
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It seems improbable, at first, that signaling roles of PIP2 might be discovered only now, after the biochemical reactions just described have been studied for many years. In fact, the potential complexities of PIP2-dependent signaling are daunting. First, PIP2 changes may occur in a highly localized fashion. In the extreme case, PIP2 might be generated and metabolized directly at its binding sites, as occurs for GTP in G protein signaling (21). Second, even if PIP2 levels change, it may be difficult to differentiate effects of PIP2 changes from effects of its metabolites. Third, there are no reliable pharmacological tools to inhibit individual mechanisms involved in PIP2 metabolism. Fourth, quantitative measurements of PIP2 are more problematic than measurements of IP3. One recent innovation, which overcomes some of these problems, is to monitor the membrane association of green fluorescent protein-coupled PIP2-binding domains (57, 62). However, the domains employed bind IP3 with much higher affinity than they bind PIP2, so they can be used with equal validity to monitor IP3 changes in cells (30). Also, the application of these probes is limited because they must be expressed in cells.
For cardiac muscle, still another complexity is that
phosphatidylinositide signaling appears to be altered by isolation of cardiac myocytes. In intact heart, total PIP2 levels amount
to 10-30 µmol/kg wet weight (47, 72), and
IP3 levels are in this same range (120-300 pmol/mg
protein or 12-30 µmol/kg wet weight; see references listed in
Ref. 72). PIP2 and IP3 levels both plummet in response to cell isolation, and they remain depressed in
cultured adult myocytes (71, 72). An abundance of studies, in which neonatal and adult myocyte cultures were used, demonstrate increases of inositol phosphates, including IP3, with
activation of Gq-coupled receptor pathways
(72). However, basal IP3 levels are so high in
intact heart that it is difficult to demonstrate a rise of
IP3 with activation of G
q-coupled receptors.
In rat ventricle, a rise of IP3 can be demonstrated with
strong
-adrenergic stimulation by phenylephrine (23)
but not in response to the physiological agonist norepinephrine
(50). In rat atrium, generation of IP3 is not
obviously increased during the norepinephrine response, and some
results suggest that inositol phosphates are being generated from
sources besides PIP2 (71, 73).
In short, it is still not clear whether PIP2 changes
significantly during physiologically relevant cell signaling changes in
cardiac muscle. In tissue slices prepared from canine atrial muscle,
PIP2 levels fall by 20-25% within a few seconds
during strong muscarinic receptor stimulation, and they return to
baseline within 30 s with continued agonist application,
accompanied by a Ca2+-dependent increase of PI synthesis
(55). In superfused right ventricular strips from rat,
strong -adrenergic stimulation results in a modest decrease of
PIP2 with a smaller rebound in a few minutes; during a
rapid train of electrical stimuli, IP3 levels rise by about
30% whereas PIP2 levels do not change (53).
In addition to these reports, it has been suggested that
PIP2 levels may increase during
-adrenergic stimulation
in intact cardiac tissue (13, 33).
Given this paucity of information, it seemed important to reexamine effects of major cardioactive hormones on PIP2 in heart. Also, it seemed important to begin to examine the influence of other physiologically relevant processes on cardiac PIP2. At the same time, however, it seemed very unattractive to perform such studies with conventional isotope flux techniques, especially with the use of intact cardiac tissue, because the measurements require long labeling times and high specific activities that are simply not practical for repeated measurements in such tissue. The alternative, then, is to perform some type of mass measurements of phospholipids that do not require radiolabeling, and the application of a mass spectrometric method is the obvious first choice. To date, however, mass spectrometric methods have not proved useful to sensitively and quantitatively detect the more negatively charged, low-abundance phospholipids, such as PIP2. Therefore, we developed and applied a new HPLC method that is highly quantitative above moderately sensitive detection limits in the range of 30 pmol (47). In brief, the total masses of anionic phospholipids from tissue samples are quantified by suppressed conductivity measurements after deacylation of the phospholipids and separation of the head groups by anion exchange HPLC. Besides examining G protein-coupled hormone responses in intact heart, we have identified substantial effects of contraction frequency, osmolarity, and DAG analogs on cardiac PIP2, and we supplement these measurements with comparative measurements in standard cell lines.
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METHODS |
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Cardiac myocytes and giant patch recording.
Guinea pigs (250-600 g) were killed by intraperitoneal injection
of 10 mg/kg pentobarbital sodium. Hearts were removed after loss of reflexes and perfused in retrograde fashion (2.5 ml · g1 · min
1) at 37°C
with a solution containing 140 mM NaCl, 5 mM KCl, 10 mM HEPES, 0.5 mM
Na2HPO4, 1.2 mM CaCl2, 0.5 mM
MgCl2, 2 mM taurine, and 15 mM glucose, adjusted to pH 7.4 with NaOH and saturated with oxygen. Cardiac myocyte preparation and
storage and Na/Ca2+ exchange current recording were carried
out as described previously (10).
Atrial contraction. Left atria of young (<400 g) guinea pigs were harvested before preparation of ventricular myocytes. The atria were mounted in a water-jacketed (35°C) gas-lift superfusion chamber that recirculates bath solutions rapidly directly past the muscle. The perfusion solution described was used as bath solution, the atria were electrically stimulated at 2× threshold (1 ms), and contractions were recorded isometrically.
Measurements of anionic phospholipids. Anionic phospholipids were quantified from quick-frozen heart tissue samples and from cell cultures as described previously (47). Briefly, after retrograde perfusion of guinea pig hearts began, the atria were dissected away and the left atria were cut in two roughly equal pieces. The atrial halves were then maintained in two open, temperated (35°C) water baths that recirculated oxygenated perfusion solution directly past the muscles. Before being frozen, atrial halves were held at their edge with fine tweezers and touched to a punctate platinum electrode in the solution path to allow electrical stimulation at 2× threshhold with 1-ms duration. After completion of a protocol, the tissue sample was frozen within 0.3 s in metal clamps (3 × 3× 0.5 cm) that were precooled in liquid nitrogen. Ventricular tissue biopsies of 70-140 mg were quickly cut from the retrograde-perfused guinea pig ventricles (37°C) and frozen within 1 s by being pressed (or smashed) between metal blocks or clamps precooled in liquid nitrogen. In preliminary experiments, we verified that multiple samples could be taken from one heart with very little variability or time-dependent changes. Both PIP and PIP2 levels tended to decrease with perfusion time, and in six hearts the average decrease measured in consecutive samples was 15 ± 5% over 45 min. Duplicate phospholipid determinations were made from each ventricular biopsy, and the results were averaged. The atrial samples allowed only one determination per tissue sample.
Phospholipids were extracted from pulverized tissue in 1 ml of cold (less thanCell cultures. COS, HEK-293, HeLa, and M1 (34) cell cultures were grown to confluence in 10-cm dishes with the use of standard culture medium for each cell type, and phospholipids were extracted for measurement of anionic phospholipids as described previously (47).
Materials. All materials were obtained from sources given previously (47). Pleckstrin homology (PH) domains were expressed and isolated as described (15, 35).
Statistical analysis. Experimental results presented are the averages of three or more tissue samples, in most cases four to seven. All error bars represent standard errors (SE) of the mean. Results are presented as mole percent of total anionic phospholipid (i.e., percent of total anionic phospholipids calculated on a mole basis). This quantification is less variable than wet (frozen) weight, because the weight determinations include variable amounts of water. Statistical significance was determined by Student's unpaired t-test and by Student's t-test for paired observations when comparing results from the same heart.
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RESULTS |
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Determinants of PIP2 in cardiac sarcolemma: impressions
from PIP2-sensitive currents.
To understand how cardiac PIP2 is regulated, we must
ultimately be able to coordinate measurements of PIP2 in
intact cells with the regulation of PIP2-sensitive
transport mechanisms and with biochemical studies of PIP2
metabolism in isolated membranes. As orientation, we present in Fig.
2 typical current measurements in
inside-out cardiac sarcolemmal patches that suggest simple kinetic and
mechanistic conclusions about the generation and breakdown of
PIP2. The activation of Na+/Ca2+
exchange current by cytoplasmic ATP, described in Fig. 2, evidently tracks the generation of PIP2, which binds to and activates
the exchanger by suppressing an autoinhibitory inactivation process (24, 26). For clarity, the presumed changes of
PIP2 are depicted qualitatively below the current records
in Fig. 2. The results presented are typical outward exchange currents
(i.e., Ca2+ influx exchanger mode; 37°C). As shown in
Fig. 2A, the exchange current was initially
activated by substituting 60 mM Cs+ on the cytoplasmic side
for 60 mM Na+; 2 mM Ca2+ was present in the
pipette. The current decayed by about 80% over a few seconds, which
reflects the function of the exchanger's inactivation domain, and the
current would remain stable at this low level without application of
ATP. With application of 2 mM ATP, the current increased within 30 s to a magnitude that is somewhat greater than the peak current
obtained upon application of cytoplasmic Na+, and the
current usually remained stable at this activated level for a few
minutes after removal of ATP when the cytoplasmic free Ca2+
level was as low as it is here (0.5 µM). During that time period, two
recombinant PH-domains were applied and removed from the cytoplasmic side at concentrations of 2 µM. The first domain is the GRP1 PH domain that binds PIP3 with much higher affinity than it
binds PIP2 (35). It was without significant
effect at this concentration. The second domain is the PLC- PH
domain (15). It inhibited the exchange current within
seconds to the baseline steady-state level recorded before application
of ATP, and the inhibitory effect of the domain could be
washed out quickly, consistent with a relatively low PIP2
affinity. The PLC-PH domain has a dissociation constant for
PIP2 of about 1 µM . Thus it is reasonable that, at a
concentration of 2 µM, the domain can mask the stimulatory effect of
ATP for the most part. PIP2 antibodies (31)
inhibit the Na+/Ca2+ exchange current to a
similar extent as the PLC-PH domain, but the effects of antibodies do
not wash out, indicative of much tighter binding to PIP2
(data not shown). Also, high concentrations of wortmannin (2 µM) have
no evident effect on the stimulation of
Na+/Ca2+ exchange current, or K+
currents, by ATP in cardiac patches. This is consistent with the idea
that the major PI kinase in the patch is a wortmannin-insensitive, type
2 PI 4-kinase (2).
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PIP2 in atrial muscle.
We first tested for changes of cardiac PIP and PIP2 in
response to several agonists that are known to activate PLCs in heart via Gq-coupled pathways, starting with muscarinic
stimulation in atrial muscle. The acetylcholine-sensitive
K+ (GIRK)-channels in atrial muscle are activated by
release of
-subunits from G
i upon acetylcholine
binding at M2-muscarinic receptors (43). In
heart, as well as other tissues, the activation of GIRKs can be
dampened by the parallel activation of G
q/PLC-coupled pathways (29, 36). Depletion of PIP2 by PLC
activity is one possible explanation for this inhibition, as well as
for the fade or "desensitization" of acetylcholine responses
(36). Figure 3,
A-C, summarizes our relevant results for superfused
left guinea pig atria, in which we examined effects of strong
muscarinic receptor activation by the stable agonist carbachol.
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PIP2 in ventricle.
Figure 4 describes effects of activating
four G protein-coupled receptor systems in beating, arterially perfused
guinea pig ventricles. As shown in Fig. 4A, stimulation of
-adrenergic receptors by 50 µM phenylephrine for 10 min had no
effect on PIP2 and caused a significant (24%) increase of
PIP. Similarly, as shown in Fig. 4B, stimulation of
endothelin receptors by 0.5 µM endothelin-1 for 10 min had no
significant effect on PIP2 or PIP, although PIP tended to
increase. Adenosine activates multiple receptors in most cardiac
tissues and is thought to be a major mediator of protein kinase C (PKC)
activation in cardiac ischemia (8). Because
adenosine has strong negative chronotropic effects, hearts were paced
electrically via punctate platinum electrodes at 2 Hz inserted into the
ventricular wall. As shown in Fig. 4C, application of 0.2 mM
adenosine for 10 min caused significant (22%) depletion of
PIP2 without a significant change of PIP, and this is the
only significant agonist-induced PIP2 depletion that we
have identified to date. Figure 4D shows the responses
obtained for strong activation of cardiac
-receptors by 0.5 µM
isoproteronol for 10 min, which, on the basis of visual inspection,
induced large increases of contraction magnitude and frequency. In
contrast to a previous report (33), PIP2 did
not change in guinea pig ventricles, whereas PIP increased
significantly by 19%. Similar experiments with guinea pig atria
generated very similar results (data not shown).
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DAG-dependent mechanisms can cause PIP2 depletion in
heart.
The results presented up to now demonstrate that stimulation of several
PLC-coupled receptor systems does not result in global PIP2
depletion, at least not when agonists are applied one at a time. Two
explanations are possible. Hormone-activated PLC activity may not be
active enough to deplete PIP2 significantly, or else compensatory mechanisms ensure that PIP2 does not decrease
in intact cells. A role for compensatory mechanisms is supported by the
observation that PIP levels increase with several of the agonists
employed, and one simple feedback possibility is that PKC activation by
DAG results in the activation of lipid kinases. Evidence for such a
mechanism has been described previously in multiple cell types
(5, 22, 48). Therefore, we tested for effects of strong
PKC activators on PIP2 in cardiac tissues and, for
comparison, in cell cultures. As shown in Fig.
5A, 12-min treatment with the
phorbol ester, phorbol 12-myristate 13-acetate (PMA; 1 µM), caused
depletion, not enhancement, of PIP2 by >30%. Both PIP and
PI (not shown) increased on average, but the changes were not
significant. In another group of experiments in ventricles, with the
use of a lower PMA concentration (0.5 µM), PIP2
significantly decreased by 14%, whereas PIP significantly increased by
9% (data not shown).
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PIP2 is activity dependent in heart.
After testing hormone-coupled PLC-activation, we tested other
interventions that might affect cardiac PIP2 by other
pathways, starting with changes of electrical pacing. As shown in Fig.
6, a decrease of contractile activity
caused by decreasing contraction frequency led to significant reduction
of total PIP2 in several isolated heart preparations.
Figure 6, A-D, shows results for isolated, paired left
guinea pig atria, which are completely quiescent in the absence of
electrical stimulation. The results in Fig. 6A are for
atrial halves that were initially stimulated at 0.1 Hz for 15 min.
Thereafter, one atrial half was quick-frozen, and the second half was
stimulated at 2.0 Hz for 5 min before freezing. PIP2 levels
were increased by 26% at 2.0 Hz (P < 0.01), whereas PIP levels (not shown) did not significantly change. Figure
6B shows results from a reverse protocol. First, the atrial
halves were left unstimulated for 15 min, and then both halves were
stimulated at 2.0 Hz for 5 min, one half was quick-frozen, and
electrical stimulation to the other half was terminated for 5 min
before freezing. PIP2 was decreased by 29%
(P < 0.01) in the quiescent atria compared with the
electrically stimulated atrial halves. Thus the effect of electrical
stimulation is reversible on the time scale of 5 min. Again, PIP did
not change significantly (data not shown).
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PIP and PIP2 levels are highly sensitive to cell volume changes. We examined effects of cell volume changes on PIP and PIP2 for three reasons. First, cell volume changes affect numerous regulatory processes by changing the concentrations of cell constituents, by mechanically perturbing cytoskeleton, and by affecting membrane tension. Resolution of changes to such a nonspecific intervention may in the long run provide paradigms to probe specific regulatory mechanisms. Second, some of the ion channels and transporters that are affected by cell volume changes are candidates for regulation by PIP2. Both Na+/H+ exchange (20) and Na+/Ca2+ exchange (74) are PIP2 sensitive, and both are activated by shrinkage. Third, the molecular mechanisms by which volume changes affect ion transport are still not well established.
To test for effects of modest osmolarity changes in the hypotonic range, we compared PIP and PIP2 levels in guinea pig ventricles perfused with a solution containing reduced total NaCl (100 mM) vs. the same solution with 100 mM added sucrose. Six ventricles were perfused first with the hyposmotic solution for 10 min, and then a tissue sample was taken, the ventricle was perfused for 5 min with the sucrose-containing solution, and a second sample was taken. In six other ventricles, the order of the experiment was reversed. Regardless of the order, PIP2 and PIP were increased by about 20% in the solution with higher osmolarity, and Fig. 7A presents the pooled results.
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DISCUSSION |
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In this study we have characterized total PIP and PIP2 levels in intact cardiac tissue in relation to several physiologically relevant interventions, including activation of G protein-coupled receptor systems, electrical pacing, and changes of cell volume. The measurements have two definite limitations. First, cardiac tissue contains multiple cell types, although myocytes certainly make up the majority of tissue volume. Second, PIP2-dependent signaling may take place in a localized fashion within cells so that measurements of global phosphoinositides will miss important signaling events entirely. Nevertheless, the measurements provide important clues about factors that may regulate cardiac PIP2. Changes of PIP2 on the order of 30% (Figs. 5A, 6A, and 7) are sufficiently large to affect some PIP2-sensitive mechanisms. For example, the high PIP2 concentrations of intact heart, compared with isolated myocytes (47), will presumably support KATP channel opening in the presence of relatively high ATP concentrations (61). A 30% increase of PIP2 with increase of cardiac frequency, as might occur during vigorous exercise, would presumably cause the opening of a small fraction of KATP channels as a physiological regulatory mechanism, independent of metabolic inhibition. Furthermore, it can be assumed that local changes of PIP2 are larger than the global changes actually measured.
Control of cardiac PIP2.
Cardiac myocytes, like most cells, contain multiple PLC types that are
modulated by multiple cell signaling pathways, including not only
Gq-coupled receptors but also tyrosine kinases, probably trafficking mechanisms, and Ca2+ itself (67).
Three agents that with good certainty activate G
q /PLC-coupled pathways
carbachol,
phenylephrine, and endothelin-1
do not cause PIP2
depletion in guinea pig atria or ventricles. Of course, we might have
missed large local changes in these measurements, and depletion may be
masked by biochemical mechanisms that favor resynthesis of
PIP2 (for example, see Refs. 54 and 63). As already noted in connection with Fig. 2, our failure to resolve G
q/PLC-mediated PIP2 depletion is consistent
with our failure, over several years, to find evidence for
G
q-mediated inhibition of PIP2-dependent
currents in cardiac membrane patches. Whether other hormones, such as
insulin, might activate more powerful PLC systems, or whether we have
failed to properly activate G
q-coupled PLC's in
patches, remains to be seen. Patch recordings described here (Fig. 2)
do indeed favor the idea that the Ca2+-dependent activation
of one or more PLCs can deplete PIP2 in the cardiac
sarcolemma very rapidly, and our results for electrical stimulation in
the presence of isoproterenol (Fig. 6D) confirm that
PIP2 can decrease during trains of action potentials when Ca2+ transients are of large magnitude (53).
If the patch results are relevant to the intact cells, then the
Ca2+ affinity of the PLC involved is so low that it could
only sense Ca2+ transients close to Ca2+
release sites.
Effects of DAG analogs.
The suggestion that PKCs might activate the synthesis of PIP and
PIP2 in close association with the activation of
Gq/PLC-coupled pathways was made several years ago
(5, 22), and our results do not contradict this
possibility. One possible site of action is the phosphorylation of
PI-transfer proteins by PKCs (68). However, for
PMA application in heart and in the M1 kidney cell line, depletion of
PIP2, rather than enhancement, can be of substantial magnitude. Possibly, this PIP2-depleting effect supports
the inhibition of inward rectifier K+ channels that is
often observed with PKC activation (19, 29, 40, 44, 69)
and that in some cases is known to involve channel phosphorylation
(40). Because both PIP and PI tend to increase when
PIP2 decreases in the presence of DAG analogs, the results are consistent with an activation of lipid phosphatases. DAG-dependent mechanisms affect profoundly both insertion and retrieval processes at
the surface membrane, and in yeast some lipid phosphatases are
regulated by trafficking mechanisms (49). Thus we
speculate that a DAG-dependent trafficking mechanism may regulate
cardiac lipid phosphatases.
Volume sensitivity of PIP and PIP2. In the absence of a rigid cell wall, all cells must regulate their cell volume in the long-term, and numerous regulatory mechanisms are likely involved. However, no pathway is firmly established from sensor to effector at this time. Changes of PIP and PIP2 with changes of cell volume (Figs. 7 and 8) are large, robust, rapidly reversible, and highly reproducible responses of cardiac muscle and several cell lines. Also, in plants, PIP2 increases during hyperosmotic stress (51). The responses are not blocked by inhibiting a number of signaling mechanisms that are known to be changed during cell shrinkage, including the disruption of microtubules. Possibly, therefore, the responses rely on a relatively direct mechanism, and one speculation is that the insertion of membrane that can occur with cell swelling (37) brings important regulatory enzymes to the surface membrane. Because both PIP and PIP2 change with osmolarity changes, insertion of lipid phosphatases in hypotonic solutions, as well as their retrieval in hypertonic solutions, would account for the results. Regardless of the molecular mechanism, our failure to pharmacologically influence the effects of shrinkage are reminiscent of attempts to pharmacologically influence the activation of Na+/H+ exchange activity by cell shrinkage (20). The stimulation of cardiac Na+/Ca2+ exchange by cell shrinkage also does not appear to involve either protein kinases or microtubules (74). It seems reasonable, therefore, to suggest that the shrinkage-induced increase of PIP2 may activate both of these transport systems in hypertonic medium.
In summary, whole tissue measurements of PIP and PIP2 demonstrate that G ![]() |
ACKNOWLEDGEMENTS |
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We thank Drs. Dong M. Kang, Helen Yin, and Joe Albanesi (University of Texas Southwestern) for helpful discussions and encouragement.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-515323 (to D. W. Hilgemann).
Address for reprint requests and other correspondence: D. W. Hilgemann, Dept. of Physiology, Univ. of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9040 (E-mail: hilgeman{at}utsw.swmed.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.
First published April 18, 2002;10.1152/ajpcell.00486.2001
Received 12 October 2001; accepted in final form 20 February 2002.
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