(Received for publication, July 12, 1995; and in revised form, October 4, 1995)
From the
Anisomycin or osmotic stress induced by sorbitol activated c-Jun N-terminal protein kinases (JNKs) in ventricular myocytes cultured from neonatal rat hearts. After 15-30 min, JNK was activated by 10-20-fold. Activation by anisomycin was transient, but that by sorbitol was sustained for at least 4 h. In-gel JNK assays confirmed activation of two renaturable JNKs of 46 and 55 kDa (JNK-46 and JNK-55, respectively). An antibody against human JNK1 immunoprecipitated JNK-46 activity. Endothelin-1, an activator of extracellular signal-regulated protein kinases (ERKs), also transiently activated JNKs by 2-5-fold after 30 min. Phorbol 12-myristate 13-acetate did not activate the JNKs although it activated ERK1 and ERK2, which phosphorylated the c-Jun transactivation domain in vitro. ATP depletion and repletion achieved by incubation in cyanide + deoxyglucose and its subsequent removal from the medium activated the ERKs but failed to activate the JNKs. Sorbitol (but not anisomycin) also stimulated the ERKs. Sorbitol-stimulated JNK activity could be resolved into three peaks by fast protein liquid chromatography on a Mono Q column. The two major peaks contained JNK-46 or JNK-55. These results demonstrate that cellular stresses differentially activate the JNKs and ERKs and that there may be ``cross-talk'' between these MAPK pathways.
The mammalian ventricular myocyte is a terminally differentiated
cell that responds to neurohumoral or mechanical stimuli by
adaptational growth in the absence of cell division
(hypertrophy)(1) . Increases in cell size and myofibrillar
content are accompanied by transcriptional changes such as the rapid
but transient expression of the immediate-early genes c-fos,
c-jun, and egr-1 that encode transcription
factors(1) . Expression of c-fos has been used as an
early marker of the hypertrophic response in the heart (2) and
is controlled by multiple promoter elements that include the serum
response element, the sis-inducible element, the activator
protein-1 element (AP-1), ()and the cAMP response
element(3) . Regulation via the serum response element involves
the phosphorylation of the ternary complex factor p62
(or
Elk-1) by the extracellular signal-regulated protein kinase (ERK)
subfamily of mitogen-activated protein kinases
(MAPKs)(
)(4) , whereas the sis-inducible
element is involved in receptor protein tyrosine kinase
signaling(3) . Once expressed, Fos and other members of the
Fos-related family of transcription factors heterodimerize with members
of the c-Jun family and transactivate at promoter regions containing
AP-1 sites(5) .
Rapid changes in transcriptional activity also follow the phosphorylation of c-Jun on Ser-63 and Ser-73 in its N-terminal transactivation domain(6, 7) . Although ERKs will phosphorylate Ser-63 and Ser-73, another protein kinase, pp54, also phosphorylates these residues(7) . This Ser/Thr protein kinase has been identified as a member of a c-Jun N-terminal kinase (JNK) subfamily of MAPKs(8) . JNKs are only weakly activated by growth factors or phorbol esters, but are strongly activated by inflammatory cytokines and cellular stresses such as UV radiation, heat shock or protein synthesis inhibition (9, 10, 11) .
We recently proposed an
involvement of the Ras Raf
MEK
ERK pathway in the
development of hypertrophy of the ventricular myocyte (12, 13) . This has been supported by studies in which
transfection of ventricular myocytes with
[Val
]Ha-Ras(14) , active forms of c-Raf (15) or constitutively activated forms of MEK (16) produced changes in gene expression that were
qualitatively similar to those observed after treatment with
hypertrophic agonists (1) . Transfection of myocytes with a
dominant-negative form of ERK1 prevented these changes in gene
expression induced by the hypertrophic agonist
phenylephrine(17) .
The ventricular myocyte must also adapt hypertrophically to cellular stress, such as myocardial ischemia, or reperfusion of an ischemic region(18, 19) . Although myocytes in the affected region may be irreversibly damaged and subsequently die, myocytes bordering on the damaged zone increase in size to maintain the overall contractile capacity of the heart(18, 20) . Given that the transcriptional activation observed during the hypertrophic response could involve activation of growth-related protein kinases additional to ERKs, we have examined the activation of isoforms of JNKs and ERKs in cultured neonatal rat ventricular myocytes exposed to cellular stresses or hypertrophic neurohumoral stimuli.
The phosphorylation of GST-c-Jun(1-135) by
protein kinases associated with the
GST-c-Jun(1-135)/glutathione-Sepharose pellet was performed
essentially as described(10) . The phosphorylation reaction was
initiated with 30 µl of Kinase Buffer (20 mM HEPES, 20
mM MgCl, 20 mM
-glycerophosphate, 2
mM DTT, 0.1 mM Na
VO
, pH 7.6)
containing 20 µM ATP and 1-2 µCi of
[
-
P]ATP. After 20 min at 30 °C, the
reaction was terminated by centrifugation and washing the pellet with
cold Buffer B containing 0.05% (v/v) Triton X-100. Phosphorylated
proteins in the pellet were eluted with boiling SDS-PAGE sample buffer (29) and separated by SDS-PAGE. Following staining with
Coomassie Blue to detect the 46-kDa GST-c-Jun(1-135) and
autoradiography,
P incorporation into
GST-c-Jun(1-135) was determined by Cerenkov
counting.
Exposure of ventricular myocytes to 0.5 M sorbitol or 50 ng/ml anisomycin produced 2-5-fold changes in JNK activity after 5 min, and this increased to 10-20-fold changes after 15-30 min (Fig. 1A). Activation of JNK by these agonists was also observed in cells that had not been serum-starved (results not shown). ET-1 (100 nM) activated JNK, but maximal activation (5-fold) was less than with sorbitol or anisomycin (Fig. 1A). PMA (1 µM) did not activate JNK (Fig. 1A). Pretreatment with PMA (1 µM, 24 h), which depletes the classical (c) and novel (n) PKC isoforms of these cells (13) , did not affect activation of JNKs by sorbitol (results not shown), indicating a cPKC/nPKC-independent pathway of activation.
Figure 1:
Activation of JNKs in
cultured neonatal rat ventricular myocytes. A, time course for
the activation of JNK. Individual 35-mm dishes of ventricular myocytes
were exposed to 0.5 M sorbitol (), 50 ng/ml anisomycin
(
), 1 µM PMA (
), 100 nM ET-1
(
), or serum-free control medium (
). Extracts were assayed
for JNK activity using the solid-phase JNK assay described under
``Experimental Procedures.'' JNK activities (mean ±
S.E., n = 3-8 independent observations, except
for 120, 180, and 240 min where n = 2) are expressed
relative to the activities in untreated cells. B, comparison
of the effect of sorbitol and ET-1 on JNK activation. Individual 35-mm
dishes of ventricular myocytes were exposed to 0.5 M sorbitol
or 100 nM ET-1. Extracts were assayed for JNK activity by the
in-gel protein kinase assay using 0.1 mg/ml GST-c-Jun(1-135)
polymerized in 10% SDS-PAGE gels as described under ``Experimental
Procedures.'' The molecular masses (kDa) of marker proteins are
indicated by the numbers to the left of the panel.
The arrows indicate the positions of the 55- and 46-kDa JNKs
(JNK-55 and JNK-46, respectively).
The time
course of JNK activation was examined (Fig. 1A).
Activation of JNK by sorbitol was sustained during a 4-h exposure (Fig. 1A). This activation following osmotic shock
contrasted with the transient activation of JNK following osmotic shock
of HeLa cells (32) or the exposure of other cell types to UV
stress(8) , carbachol (33) ,
interleukin-1(32) , or tumor necrosis
factor-(34) . Activation of JNK in myocytes by anisomycin
or ET-1 was transient, being reversed after 180-240 min (Fig. 1A).
The protein kinases that phosphorylate GST-c-Jun(1-135) were characterized by in-gel kinase assays. No activity was detected in extracts of untreated cells (Fig. 1B) or when GST-c-Jun(1-135) was omitted from the gels (results not shown). Following exposure to sorbitol or ET-1 for 5 min, two protein kinases with molecular masses of 46 kDa (JNK-46) and 55 kDa (JNK-55) were detected (Fig. 1B). The JNK-55 band was less intense than JNK-46. Activation of both JNKs followed a similar time course and, in agreement with solid-phase JNK assays (Fig. 1A), the activation by sorbitol was at least 3-fold greater than the activation by ET-1. The molecular masses of these JNKs agree with those for renaturable JNKs previously described(8, 10, 35) .
Total extracts of ventricular myocytes exposed to 1
µM PMA were examined by in-gel kinase assays using either
GST-c-Jun(1-135) (Fig. 2A) or MBP (Fig. 2B). PMA activated two protein kinases of 42 and
44-46 kDa when either MBP or GST-c-Jun(1-135) was the
substrate. The ability of the 42- and 44-46-kDa protein kinases
to phosphorylate MBP (Fig. 2B) suggests that ERK1 and
ERK2 rather than JNKs are responsible for the GST-c-Jun(1-135)
phosphorylation. The finding that PMA did not activate JNKs using
solid-phase assays (Fig. 1A) supports this conclusion.
However, when total extracts of PMA-treated myocytes were incubated
with GST-c-Jun(1-135) and [-
P]ATP,
GST-c-Jun(1-135) phosphorylation was detectable (results not
shown), confirming that PMA-activated non-JNK GST-c-Jun(1-135)
kinases are present in extracts. Others have shown that ERKs copurify
with phorbol ester-activated c-Jun kinases (41) , supporting
the conclusion that both ERKs and JNKs phosphorylate the
transactivation domain of c-Jun.
Figure 2: PMA activates ERK1 and ERK2 but anisomycin activates JNK-46 and JNK-55. Individual 35-mm dishes of ventricular myocytes were exposed to 1 µM PMA or 50 ng/ml anisomycin for the times indicated at the bottom of each panel. Extracts were assayed by in-gel kinase assays using 0.1 mg/ml GST-c-Jun(1-135) (A) or 0.5 mg/ml MBP (B) polymerized in 10% SDS-PAGE gels as described under ``Experimental Procedures.'' Each gel is representative of two independent experiments. The molecular masses (kDa) of marker proteins are indicated by the numbers to the left of each panel. The arrows indicate the positions of the protein kinase activities that change upon exposure to PMA or anisomycin. In A, these migrate at 55, 44-46, and 42 kDa; in B, they migrate at 44-46 and 42 kDa.
It has been suggested that PMA
activates JNKs in T cells only when Ca influx is
stimulated. Thus, PMA and the Ca
ionophore A23187
synergistically stimulate JNK activity(35) . This finding could
be relevant to our failure to observe activation of JNKs by PMA (Fig. 1A and 2A). However, simultaneous
exposure of ventricular myocytes to PMA (1 µM) and the
Ca
ionophore, ionomycin (0.1 µM) did not
activate JNKs as assayed by in-gel GST-c-Jun(1-135) kinase assays
(results not shown).
In contrast to PMA, anisomycin (50 ng/ml) activated JNK-46 and JNK-55 (Fig. 2A). The JNK-55 band was less intense than that for JNK-46. Anisomycin only weakly activated ERK1 and ERK2 as shown by in-gel phosphorylation of MBP (Fig. 2B).
Figure 3: Activation of ERKs and JNKs by sorbitol and phenylephrine. Individual 35-mm dishes of ventricular myocytes were exposed to serum-free medium (CON), 0.5 M sorbitol (SRB), 50 ng/ml anisomycin (AN), 1 µM PMA, or 50 µM phenylephrine (PE) in serum-free medium (DMEM/M199) for 30 min. Extracts were prepared then resuspended in SDS-PAGE sample buffer (panels A and B) or subjected to the solid-phase JNK assay up to the glutathione-Sepharose precipitation step (panel C) as described under ``Experimental Procedures.'' The pellets from the solid-phase assay glutathione-Sepharose precipitation step (panel C) were then resuspended in SDS-PAGE buffer. A, activation of MBP kinases was assayed by the in-gel kinase method using 0.5 mg/ml MBP polymerized in 10% SDS-PAGE gels as described under ``Experimental Procedures.'' B and C, activation of JNKs was assayed by the in-gel kinase method using 0.1 mg/ml GST-c-Jun(1-135) polymerized in 10% SDS-PAGE gels as described under ``Experimental Procedures.'' Each gel is representative of two independent experiments. The molecular masses (kDa) of marker proteins are indicated by the numbers to the left of each panel. The arrows indicate the positions of the protein kinase activities that change upon exposure to sorbitol, anisomycin, PMA, or phenylephrine. In A, these migrate at 44-46 and 42 kDa; in B, they migrate at 55, 44-46, and 42 kDa; in C, they migrate at 55 and 44-46 kDa.
As described above, PMA activated two GST-c-Jun(1-135) kinases that appeared to correspond with ERK1 and ERK2 (Fig. 3, compare A and B). These kinases were not recovered in the GSH-Sepharose pellet following interaction with GST-c-Jun(1-135) (Fig. 3C), but remained in the supernatant (results not shown). This is consistent with the weaker interaction of ERKs with the N terminus of c-Jun(31) . Using PMA-activated GST-c-Jun(1-135) kinases as markers, it can be concluded that the weaker activation of a GST-c-Jun(1-135) kinase at about 42 kDa by sorbitol (Fig. 3B) may be attributable to ERK2. Any GST-c-Jun(1-135) kinase activity stimulated by sorbitol attributable to ERK1 is obscured by activation of JNK-46 (Fig. 3B). Phenylephrine (50 µM) only very weakly activated GST-c-Jun(1-135) kinases (Fig. 3B) and again, based on their migration relative to PMA-stimulated GST-c-Jun(1-135) kinases, these activities were probably attributable to ERK1 and ERK2. However, weak activation of JNK1 and JNK2 by phenylephrine was detected by in-gel phosphorylation of GST-c-Jun(1-135) following precipitation of JNKs interacting with GST-c-Jun(1-135) with GSH-Sepharose (Fig. 3C).
Figure 4: Immunoprecipitation of activated JNK by an anti-JNK1 antibody. Individual 60-mm dishes of ventricular myocytes were exposed to control serum-free medium, 0.5 M sorbitol (SRB), 50 ng/ml anisomycin (AN), 1 µM PMA, 50 µM phenylephrine (PE), or 100 nM ET-1 for 30 min. Extracts were prepared, then subjected to immunocomplex GST-c-Jun(1-135) kinase assays using an agarose-conjugated anti-JNK1 antibody as described under ``Experimental Procedures.'' A, JNK activities (means ± S.E., n = 4-5 independent observations) are expressed relative to activities in control cells. B, the pellets and supernatants from the immunoprecipitation step of the immunocomplex kinase assays were resuspended in SDS-PAGE buffer and activation of JNKs was assayed by the in-gel kinase method using 0.1 mg/ml GST-c-Jun(1-135) polymerized in 10% SDS-PAGE gels as described under ``Experimental Procedures.'' The molecular masses (kDa) of marker proteins are indicated by the numbers to the left of the panel. The arrows indicate the positions of the protein kinase activities that change upon exposure to sorbitol or anisomycin. These migrate at 55 or 46 kDa.
Figure 5:
Activation of c-Jun kinases by sorbitol or
PMA. A, five 60-mm dishes of ventricular myocytes were exposed
to control serum-free medium () or 0.5 M sorbitol
(
) for 30 min. Extracts were prepared and proteins separated by
FPLC using a Mono Q column with elution by a linear NaCl gradient (dotted line). Fractions were assayed for JNK activity as
described under ``Experimental Procedures.'' The peaks of JNK
activity are labeled J1, J2, and J3. B, FPLC Mono Q column fractions from sorbitol-treated myocytes
were pooled as indicated (i-iv), concentrated and
assayed by the in-gel kinase method using 0.1 mg/ml
GST-c-Jun(1-135) polymerized in 10% SDS-PAGE gels as described
under ``Experimental Procedures''. The molecular masses (kDa)
of marker proteins are indicated by the numbers to the left of the panel. C, five 60-mm dishes of
ventricular myocytes were exposed to control serum-free medium (
)
or 1 µM PMA (
) for 5 min. Extracts were prepared and
proteins separated by FPLC using a Mono Q column with elution by a
linear NaCl gradient (dotted line). Fractions were assayed for
JNK activity.
FPLC fractions from sorbitol-treated myocytes were also assayed for MBP kinases (Fig. 6A). Only a single asymmetric peak of MBP kinase was observed (M1, Fig. 6A). Although peaks M1 and J3 (Fig. 5A) overlapped, peak J3 consistently preceded peak M1 by 1 to 2 fractions (n = 3 separate occasions, compare Fig. 5A and Fig. 6A). MBP kinases of 55, 44, and 42 kDa were detected in pooled fractions (iv) (Fig. 5B). (The autoradiograph has been overexposed to demonstrate the limited renaturable MBP kinase present in pooled fractions (i-iii)). The 55-kDa MBP kinase may possibly correspond to a JNK-55 isoform in pooled fractions (iv). The other JNK isoforms, including the 55-kDa isoform in pooled fractions (iii) and the 46-kDa isoform in pooled fractions (ii), do not utilize MBP as a substrate (Fig. 6B).
Figure 6:
Activation of MBP kinases by sorbitol or
PMA. A, FPLC Mono Q column fractions prepared from ventricular
myocytes exposed to control serum-free medium () or 0.5 M sorbitol (
) for 30 min as described in the legend to Fig. 5were assayed for MBP kinase activity. B,
FPLC Mono Q column fractions from sorbitol-treated myocytes were pooled
as indicated (i-iv), concentrated, and assayed by the
in-gel kinase method using 0.5 mg/ml MBP polymerized in 10% SDS-PAGE
gels as described under ``Experimental Procedures.'' The
molecular masses (kDa) of marker proteins are indicated by the numbers to the left of the panel. C, FPLC
Mono Q column fractions prepared from ventricular myocytes exposed to
control serum-free medium (
) or 1 µM PMA (
) for
5 min as described in the legend to Fig. 5were assayed for MBP
kinase activity.
As shown by immunoblotting of pooled fractions (iv) with anti-ERK1/ERK2 antiserum(24) , the 42- and 44-kDa MBP kinases correspond to ERK2 and ERK1, respectively (results not shown). We also separated extracts of PMA-treated myocytes by FPLC on MonoQ (Fig. 6C). Two peaks of PMA-stimulated MBP kinase activity were detected (Fig. 6C). These peaks co-elute with the PMA-stimulated c-Jun kinases (Fig. 5C). Thus, PMA appears to stimulate the ERKs in the absence of significant JNK activation.
We examined the time course of inactivation of JNK-46 in the ventricular myocyte following activation by 0.5 M sorbitol. Cells were treated with sorbitol for 15 min, and then followed by incubation with serum-free medium for 15, 45, or 60 min. Immunocomplex kinase assays (which detect principally JNK-46; Fig. 4B) demonstrated that JNK-46 was inactivated within 15-45 min of removal of sorbitol (Fig. 7). The inactivation of both JNK-46 and JNK-55 was confirmed using the in-gel GST-c-Jun(1-135) kinase assays (results not shown). These results suggest that sorbitol provides a potent and prolonged stimulation of the signaling pathway leading to activation of JNKs, and that JNKs can be rapidly inactivated after removal of the positive signaling components.
Figure 7:
Activation of JNKs by sorbitol is
reversible. Individual 60-mm dishes of ventricular myocytes were
exposed to serum-free medium or 0.5 M sorbitol for 15 min. The
medium was then aspirated and the cells exposed to either serum
free-medium (, dashed line) or 0.5 M sorbitol
(
, solid line). Extracts were prepared then subjected to
immunocomplex GST-c-Jun(1-135) kinase assays using an
agarose-conjugated anti-JNK1 antibody as described under
``Experimental Procedures.'' This method principally assays
JNK-46. JNK-46 activities (means ± S.E., n =
three independent observations) are expressed relative to the
activities in control cells.
An in vitro tissue culture model of ischemia is the inhibition of ATP synthesis by exposure of myocytes to KCN+DOG(28, 49) . Recently it has been shown that ERKs are activated during the recovery from metabolic inhibition(28) . We exposed ventricular myocytes to concentrations of cyanide and DOG previously demonstrated to deplete intracellular ATP concentrations in these cultured cells (27) . Cytosolic extracts were analyzed using the in-gel method for activation of JNKs (Fig. 8A). Neither JNK-46 or JNK-55 were activated by up to 120 min of exposure to cyanide and DOG (Fig. 8A). Furthermore, the removal of cyanide and DOG also did not activate the JNKs (Fig. 8B). Weak kinase bands of 42 and 44 kDa were detected (Fig. 8, A and B), and these appeared to correspond to the ERKs in MBP-containing gels (Fig. 8, C and D). Thus, different forms of cellular stress may activate different MAPK signaling cascades.
Figure 8: Exposure to cyanide and deoxyglucose and the subsequent removal of cyanide and deoxyglucose from the medium activates ERKs. Individual 35-mm dishes of ventricular myocytes were exposed to medium containing 1 mM KCN + 20 mM DOG for 5-120 min or to medium containing sorbitol (SRB) for 30 min. In panels B and D, the myocytes were exposed to KCN+DOG for 60 min, then exposed to fresh serum-free medium for a further 5 to 60 min. Extracts were prepared, then analyzed by the in-gel kinase method using 0.1 mg/ml GST-c-Jun(1-135) (A and B) or 0.5 mg/ml MBP (C and D) polymerized in 10% SDS-PAGE gels as described under ``Experimental Procedures.'' Each gel is representative of two independent experiments. The molecular masses (kDa) of marker proteins are indicated.
The JNKs, members of the MAPK family, are activated by phosphorylation of Thr and Tyr in a conserved Thr-Pro-Tyr sequence (reviewed in (51) ). JNK activity in the ventricular myocyte can be separated into three peaks by FPLC on Mono Q (Fig. 5A) that elute at a lower NaCl concentration than ERKs (Fig. 6, A and C). The relationship between the 44-kDa GST-c-Jun(1-135) kinase identified by FPLC on Mono Q (peak J1 in Fig. 5A) and JNK-46/JNK-55 requires further investigation. One form of the JNKs in the ventricular myocyte (JNK-46) was recognized by JNK1-specific antisera (Fig. 4, A and B). JNK-55 is presumably related to JNK-2.
Although the upstream signaling events remain unresolved, it has
become clear that diverse signals, including inflammatory
cytokines(9, 52) , protein synthesis inhibitors ( (53) and Fig. 1), ischemic reperfusion (54) ,
and osmotic stress ( (55) and Fig. 1), activate the JNK
subfamily of MAPKs. The activation of the ERK subfamily of MAPKs is
better understood and is mediated through receptor protein tyrosine
kinases or G-protein-coupled receptors(56) . In the heart, ET-1
and phenylephrine stimulate ERK activities, and this probably involves
a G-mediated activation of
PKC(13, 26, 57) . Thus PMA also strongly
activates ERKs in heart(12, 13, 38) . It was
of interest to study whether ET-1, phenylephrine, and PMA activated
JNKs, and equally whether activators of JNKs (sorbitol and anisomycin)
activated ERKs. Phenylephrine and PMA, although potent ERK activators (Fig. 2B and Fig. 3A), were the least
effective of the JNK activators tested (Fig. 1A,
2A, 3 (B and C), 4A, and
5C). Sorbitol was the most potent activator of JNKs ( Fig. 1(A and B) and 3 (B and C)) and also activated ERKs (Fig. 3A and Fig. 6(A and B)). Anisomycin was a poor
activator of ERKs in comparison to sorbitol or PMA (Fig. 2B and Fig. 3A). Interestingly, ET-1 was a moderately
effective activator of JNKs (Fig. 1A and Fig. 4A), as well as a powerful activator of
ERKs(12, 13, 26, 57) . Intracellular
signaling by ET-1 involves both G
- and
G
-coupled pathways(58, 59) . JNKs can
be activated by a G
-dependent pathway activated by the
m
-muscarinic agonist carbachol (33) or by
constitutively activated G
or G
.
The JNKs therefore can be activated by G-protein-dependent pathways in
addition to cell stress, although this may be cell- and
agonist-specific.
It is important to understand the extent of cross-talk between the JNK and ERK pathways, given that an increase in the transactivating activity of c-Jun may be an end response of activation of both pathways. Although activation of ERKs by ET-1, phenylephrine or PMA in ventricular myocytes is maximal at 5 min(13) , the maximal activation of JNKs by any agonist (including ET-1) was slower, being maximal at 30-60 min ( Fig. 1(A and B) and 2 (A and B)). If the ERK and JNK pathways are parallel as suggested (51, 60) with c-Jun as a common substrate, the mechanisms leading to differential rates of activation of each pathway must be identified. This is particularly pertinent when a single agonist (e.g. ET-1 or sorbitol) is able to activate ERKs and JNKs.
Sorbitol produced sustained activation of the JNKs in
ventricular myocytes (Fig. 1, A and B, and Fig. 7). This was not the result of an irreversible event, as
the removal of sorbitol from the medium led to rapid inactivation of
the JNKs (Fig. 7). We have been unable to detect activities of
the homologues of the osmoregulatory S. cerevisiae HOG-1
protein kinase pathway in neonatal rat ventricular myocytes. ()
This study has shown that the ventricular myocyte is able to respond to cellular stress by differential activation of the JNK or ERK pathways. The ventricular myocyte in primary culture thus serves as a useful model for studying the responses to cellular stress. The exact phenotypic consequences of the JNK activation pathway must be defined by introduction of constitutively active forms of JNKs or their upstream activators (68, 69, 70) into these cells.