(Received for publication, November 29, 1995; and in revised form, January 4, 1996)
From the
We have used reverse transcriptase-polymerase chain reaction to
investigate the expression of ryanodine receptors in several excitable
and nonexcitable cell types. Consistent with previous reports, we
detected ryanodine receptor expression in brain, heart, and skeletal
muscle. In addition, we detected ryanodine receptor expression in
various other excitable cells including PC12 and A7r5 cells. Several
muscle cell lines (BCH1, C2C12, L6, and Sol8) weakly
expressed ryanodine receptor when undifferentiated but strongly
expressed type 1 and type 3 ryanodine receptor isoforms when
differentiated into a muscle phenotype. Only 2 (HeLa and LLC-PK1 cells)
out of 11 nonexcitable cell types examined expressed ryanodine
receptors. Expression of ryanodine receptors at the protein level in
these cells was confirmed using [
H]ryanodine
binding. We also investigated the function of ryanodine receptors in
Ca
signaling in HeLa cells using single-cell Fura-2
imaging. Neither caffeine nor ryanodine caused a detectable elevation
of cytoplasmic Ca
in single HeLa cells. However,
ryanodine caused a significant decrease in the amplitude of
Ca
signals evoked by repetitive stimulation with ATP.
These studies show that ryanodine receptors are expressed in some
nonexcitable cell types and furthermore suggest that the ryanodine
receptors may be involved in a subtle regulation of intracellular
Ca
responses.
Cells have two major mechanisms available for regulating the
release of internal Ca. In one case, external signals
acting on receptors at the cell periphery generate the second messenger
inositol 1,4,5-trisphosphate, which diffuses into the cell and
mobilizes Ca
by engaging inositol 1,4,5-trisphosphate
receptors on the endoplasmic reticulum(1) . The other mechanism
employs a related, but distinct, family of intracellular channels, the
ryanodine receptors (RyRs), (
)so called because they
strongly bind the plant alkaloid ryanodine(2) . RyRs were first
identified as the intracellular calcium channels responsible for
releasing calcium from the sarcoplasmic reticulum of both skeletal and
cardiac muscle. The two striated muscle RyR isoforms have been
designated RyR1 (skeletal muscle) and RyR2 (cardiac muscle). A recently
identified isoform, referred to as RyR3, appears to be a major isoform
in brain and smooth muscle(3, 4, 5) .
Evidence for the expression of RyRs in various excitable and nonexcitable tissues has mostly been obtained using either molecular or pharmacological methods. For many excitable cells, these approaches have demonstrated the tissue-specific expression and function of RyRs ( (6) and (7) ; for a review see (8) ). However, the situation concerning the expression of RyRs in some nonexcitable tissues and their possible participation in agonist-stimulated calcium signals is far from clear(8) . In hepatocytes, for example, effects of ryanodine on agonist-induced calcium signals in intact cells (9, 10) and specific ryanodine binding sites in hepatocyte vesicles (11, 12) have been demonstrated. However, the pharmacology of the putative RyR in hepatocytes is very different from those expressed in muscle tissues (9, 13) , and furthermore, the expression of RyR mRNA in hepatocytes has not been detected using molecular techniques(7, 11, 14, 15) .
For other nonexcitable cells the converse problem exists, in that molecular techniques have identified RyR mRNA expression, but the functional evidence has been confusing, since some RyR-activating agents fail to evoke responses in these cells. For example, in mink lung epithelial cells and Jurkat T-lymphocytes, which appear to express RyR3, effects of ryanodine but not caffeine have been observed (3, 16) . In a separate study Guse et al. (17) found that Jurkat cells were caffeine-responsive.
In the present study we have examined RyR expression in a variety of tissues using RT-PCR. In addition to finding RyRs expressed in several excitable tissues and cell lines, we detected RyR expression in two nonexcitable cell types. For one of these nonexcitable cell types (HeLa cells), we investigated the function of the expressed RyRs using single-cell Fura-2 imaging.
Forward primer (RyR1,
14419-14441; RyR2, 14230-14252; RyR3, 14008-14030)
was as follows:
5`-CA(C/T)(C/T)T(A/C/G/T)(C/T)T(A/C/G/T)GA(C/T)AT(A/C/T)GCIATGGG-3`.
Reverse primer (RyR1, 14929- 14951; RyR2, 14740-14762;
RyR3, 14518-14540) was as follows:
5`-A(A/G/T)(A/G)TA(A/G)TT(A/C/G/T)GCIA(A/G)(A/G)TT(A/G)TG(C/T)TC-3`.
PCR reactions (100 µl) contained 0.4-2% of the first strand
cDNA template, 100 pmol of each primer, 0.2 mM dNTPs and 2.5
units of Taq polymerase (Boehringer Mannheim) in 10 mM Tris/HCl (pH 8.3), 50 mM KCl, and 1.5 mM MgCl. Reactions were carried out in a Biometra
UNO-Thermoblock for 30 cycles using a denaturing step for 1 min at 94
°C, annealing for 1 min at 50 °C, and extension for 1 min at 72
°C. This was followed by a final extension step at 72 °C for 10
min.
As the majority of cell types
tested were of mouse, rat, or human origin, and all three RyR isoforms
have been found in various regions of the
brain(7, 31) , we tested the primers by carrying out
PCR amplification of cDNA from mouse, rat, and human brain. A product
of the predicted size (530 bp) was amplified from all three brain cDNAs (Fig. 1A). Control reactions containing HO or
RNA were negative in the PCR (Fig. 1A). Cloning and
sequencing the brain PCR products revealed that the primers were able
to specifically amplify all three RyR isoforms. The primers were also
able to specifically amplify RyRs from mouse heart (RyR2 and RyR3) and
skeletal muscle (RyR1 and RyR3) (data not shown).
Figure 1:
RT-PCR detection of RyRs in excitable
and nonexcitable cells. RT-PCR was performed as described under
``Materials and Methods.'' Samples (one-tenth of each PCR
reaction) were subjected to electrophoresis through a 1.5% agarose gel. A, RT-PCR detection of a 530-bp fragment of different RyR
isoforms in mouse, rat, and human brain cDNA but not in control
reactions containing HO or brain RNA. B, RT-PCR
detection of RyRs in various excitable and nonexcitable cell types. The
results shown are representative of four different experiments
performed with two different RNA
preparations.
A range of cell
types was subjected to PCR amplification using these primers (Fig. 1B). Only two of the nonexcitable cell types
tested, HeLa and LLC-PK1 cells, yielded detectable PCR products. The
other nonexcitable cell types, including fibroblasts and Jurkat cells,
appeared negative in the PCR. As a positive control for the quality of
the cDNA, amplification reactions were also carried out using
-actin PCR primers. Products of the correct size and of equivalent
intensity were amplified from all cDNAs tested in this study (data not
shown).
Using the RyR primeres, products of the predicted size were
detected in most excitable cell types, including differentiated and
undifferentiated PC12 cells and A7r5 cells. A very faint PCR product
was detected in GH and Rin m5F cells, indicating that these
cell types probably also express RyRs, but very weakly. An interesting
pattern of expression was detected in the BC
H1, Sol8,
C2C12, and L6 muscle cell lines. By adjusting the culture conditions
appropriately (see ``Materials and Methods''), these cells
can either be maintained in a rapidly growing non-muscle state or
differentiated into a muscle phenotype. In the undifferentiated cells,
we detected only very weak expression of RyR by PCR. However,
differentiation of the cells correlated with the appearance of much
more abundant levels of the 530-bp product (Fig. 1B).
Fig. 2shows the restriction digest analysis of RyR PCR
products amplified from differentiated BCH1 cells,
differentiated L6 cells, and HeLa cells. The BC
H1 PCR
product was partially cut with SacI (RyR1-specific) and
completely cut with AvaII (digests RyR1 and -3) to produce
fragments of the expected sizes, suggesting that BC
H1 cells
express RyR1 and RyR3. The L6 PCR product was completely cut with SacI and not cut by either BglII (RyR2-specific) or EaeI (RyR3-specific). The HeLa cell PCR product was completely
cut with BglII (RyR2-specific) to produce fragments of the
expected size. The combined results obtained from sequencing and
restriction enzyme digest of the PCR products are presented in Table 1.
Figure 2:
Restriction enzyme digest of RyR RT-PCR
products. RT-PCR products were produced as described under
``Materials and Methods.'' PCR products were digested with
the restriction enzymes identified using the manufacturer's
suggested protocols, and the fragments produced were subjected to
electrophoresis through a 1.5% agarose gel. A, differentiated
BCH1 cells. B, differentiated L6 cells. C, HeLa cells.
Figure 3:
[H]Ryanodine binding
to total microsomal fractions isolated from various cell types.
Specific ryanodine binding was measured as described under
``Materials and Methods.'' Values are expressed in fmol of
ryanodine bound per mg of protein. Each value is the mean ± S.E.
of three to five experiments, each performed in
triplicate.
Figure 4:
Caffeine does not elevate
[Ca]
in HeLa cells.
The trace in A shows the response of a single
Fura-2-loaded HeLa cell perfused with solutions containing increasing
caffeine concentrations from 2 to 40 mM (filled
bars). Histamine (100 µM; hatched bar),
which releases Ca
from intracellular
stores(20) , was applied to show that the Ca
stores did contain releasable Ca
and to
demonstrate the typical magnitude of hormonally evoked
[Ca
]
signals. The trace is typical of 40 cells. The trace in B shows
the response of a single Fura-2-loaded HeLa cell perfused with
solutions containing caffeine (10 mM; filled bar),
ryanodine (10 µM; open bar), or caffeine (10
mM) + ryanodine (10 µM). Histamine (100
µM; hatched bar) was again added as a control.
The trace is typical of 34 cells.
The lack of caffeine effect suggested that the HeLa cells either
expressed a caffeine-insensitive RyR isoform or had a low RyR density.
We therefore altered the experimental protocol to maximize the
potential effect of the limited number of RyRs. HeLa cells were
repetitively stimulated with ATP (100 µM), which mobilizes
Ca from intracellular stores in HeLa cells (35) , either in the absence or presence of ryanodine (10
µM) (Fig. 5, A and B,
respectively). The averaged [Ca
]
values for peaks 2-7 are shown in Fig. 5, C and D. In control cells, the magnitude of the response to
repeated ATP applications decayed only slightly, probably due to
desensitization of the Ca
-signaling
pathway(35) . The magnitude of ATP-evoked responses in the
cells treated with ryanodine diminished significantly, so that the
response decayed to about 50% after six ATP applications. The latency
of the response to ATP slightly increased in the presence of ryanodine (Fig. 5E).
Figure 5:
Ryanodine attenuates Ca
responses stimulated by repetitive ATP applications. HeLa cells in
control extracellular medium were stimulated repetitively with 100
µM ATP (1-min application with 3-min wash), as shown by
the filled bars (A and B). In trace
B, 10 µM ryanodine (Ry) was present for the
time shown (open bar). The traces are representative
of 39 (A) and 63 (B) cells. The graphs in C, D, and E show the averaged Ca
release data obtained from experiments such as those shown in A and B. In C the magnitude of the response
was calculated by normalizing the area of each ATP-evoked
Ca
response relative to the area of peak 2.
In D the amplitude of each ATP-evoked Ca
response was calculated by normalizing the maximum
[Ca
]
increase during
each response relative to the height of peak 2. The graph in E shows the latency of the response to ATP (time from ATP
application to peak) in the presence and absence of ryanodine. The data
points are mean ± S.E. of 39 (
) and 63 (
) cells,
respectively. *, statistically different with p <
0.002.
The aim of the present study was to investigate the possible expression and function of RyRs in nonexcitable cells. To confirm that our PCR technique could detect RyR expression, we also analyzed several tissues known to have functional RyRs. Consistent with other studies (7, 31) , we could amplify and clone all three RyR isoforms from brain samples (Fig. 1A and Table 1). In addition, we found RyR expression in skeletal and cardiac muscle. RyR3 appeared to be expressed in both these muscle types, whereas RyR1 and RyR2 were expressed in skeletal and cardiac muscle, respectively (Table 1).
Of particular interest were
the myogenic cell lines BCH1, Sol8, C2C12, and L6, where
the intensity of the 530-bp PCR product correlated with the
differentiation of the cells from a non-muscle to a muscle phenotype.
Although the PCR method we used was not quantitative, for a limited
number of PCR cycles the intensity of the 530-bp PCR product may give
an indication of the level of RyR mRNA expression. Our observations are
consistent with several other studies linking muscle differentiation
with RyR expression(7, 23, 36) . It appears
that the myogenic cells express RyR1 (L6) or RyR1 and RyR3
(BC
H1, Sol8, and C2C12) after differentiation, similar to
the situation in skeletal muscle (7) (Table 1). Both
undifferentiated and nerve growth factor-differentiated PC12 cells were
RyR-positive (Table 1). However, there was an apparent change in
isoform expression pattern from RyR1 and RyR2 in undifferentiated cells
to only RyR1 in differentiated cells. The expression of RyR1 in
undifferentiated PC12 cells may explain the observation that
theophylline is equipotent with caffeine at stimulating Ca
release from this clone(34) , since RyR1 appears equally
sensitive to these two methylxanthines (37) .
Several
previous studies have suggested that RyR expression is growth
state-dependent; for example, aortic smooth muscle cells lose caffeine
sensitivity during their logarithmic proliferation phase, which can be
reversed or augmented by the removal or addition of growth factors,
respectively(38) . Similarly, the expression of RyR3 in mink
lung epithelial cells can be induced by exposure to
TGF-(3) . These data suggest that RyR expression can be
somewhat labile and may be associated with the state of cell
proliferation and differentiation.
After confirming that we could
detect RyR expression in these excitable tissues, we investigated the
potential expression of these receptors in 11 nonexcitable cell types,
many of which are commonly used for studies of Ca signaling (Table 1). Of these cell types, only two appeared
to express RyR mRNA. Increasing the number of PCR cycles or performing
sequential PCR reactions, using the first reaction to prime the second,
did not yield detectable products for the cell types that were
RyR-negative. The group of cell types that were found not to express
RyRs surprisingly included Jurkat T-lymphocytes (Table 1). This
result is consistent with the negligible
[
H]ryanodine binding displayed by Jurkat cells
(data not shown). These data contrast with other studies, which
suggested that RyRs were expressed in these
cells(16, 17) . The explanation for our contrasting
data is unclear but may reflect variable RyR expression between
different Jurkat cell clones.
The two RyR-positive nonexcitable cell
types, HeLa and LLC-PK1 cells, expressed RyR2 and RyR3, respectively.
RyR expression in HeLa cells was also recently reported by Giannini et al.(7) , although they identified RyR2 and RyR3 in
their control HeLa cells. The expression of only RyR2 in our HeLa cells
was confirmed by sequencing and restriction digestion of PCR products
and may again point to a clonal variation. To extend the molecular
characterization of RyR expression in these tissues, we sought to
obtain evidence for RyR at the protein level. HeLa and LLC-PK1 cells
were found to display significant levels of
[H]ryanodine binding, although it was about
17-fold less than in rabbit brain and approximately one-third of that
found in undifferentiated PC12 cells (Fig. 3).
Despite the
molecular evidence and [H]ryanodine binding data,
which clearly indicate that HeLa cells express RyRs, we were unable to
directly demonstrate a RyR-dependent
[Ca
]
increase in the cells (Fig. 4). Neither caffeine nor ryanodine evoked a measurable
increase in [Ca
]
when applied
either on their own or in combination. A similar lack of caffeine
responsiveness in HeLa cells was previously
shown(39, 40) . However, ryanodine caused a
progressive decrease in the magnitude of the Ca
signal evoked by repetitive ATP applications and a slight
increase in the latency before the
[Ca
]
rise (Fig. 5). The
decreased magnitude of the Ca
signals is consistent
with the previously described use-dependent block of RyR function by
ryanodine(34, 41, 42) , whereby the
ryanodine-bound receptors remain in a constitutively open low
conductance state. The finding that ryanodine inhibited ATP-induced
Ca
signals in a use-dependent manner suggests that
hormonal stimulation of HeLa cells brings about activation of RyRs to
amplify the normal inositol 1,4,5-trisphosphate-dependent elevation of
Ca
. Just how this channel opening is achieved is
unknown, but possibilities include activation via
Ca
-induced Ca
release (8) or production of a RyR-sensitizing ligand such as cyclic
adenosine diphosphate-ribose(43) .
The apparent paradox
between the effects of ryanodine and caffeine suggests that HeLa cells
either express a caffeine-insensitive RyR isoform or express a very low
level of RyR, such that an acute opening of the RyRs does little to
influence [Ca]
, but a prolonged
RyR opening can gradually deplete the stores. The latter explanation
seems to be the more likely, since we detected a low density of RyR in
the [
H]ryanodine binding studies (Fig. 3),
and all RyR isoforms have been shown to be
caffeine-sensitive(44) . Furthermore, similar results i.e. apparent caffeine-insensitivity and a ryanodine-induced
Ca
store depletion, have been reported by Giannini et al. (3) for mink lung epithelial cells expressing
RyR3. It seems likely that a low density of RyR may explain the lack of
effect of caffeine on [Ca
]
in
several cell types shown by molecular techniques to express
RyRs(7) .
The function of the RyRs expressed in nonexcitable
cells is not fully established. In a few cell types, such as sea urchin
eggs (29) and pancreatic acinar cells(30) , RyRs may
contribute to the initiation of Ca signals. In
hepatocytes, the recruitment of RyRs has been reported to be
agonist-specific(9) . Data from the present study suggest that
RyRs in nonexcitable HeLa cells may provide a subtle regulation of the
magnitude and kinetics (Fig. 5) of hormone-evoked
[Ca
]
responses. These data
indicate that RyRs make an important contribution to intracellular
Ca
signals in a variety of nonexcitable cell types.