From the
The rat myometrium has a rich adrenergic innervation
(1) . Activation of the adrenergic nerves modulates uterine
contractility and also subtle changes in sensitivity of the smooth
muscle to hormonal agents
(2) . Direct interaction of
norepinephrine with the
In the present study, we first
provided a molecular analysis of the
pBluescripts KS and SK with the
full-length type II cDNA (3.5 kb) and type IV cDNA (3.2 kb) adenylyl
cyclase respectively were kind gifts from Dr. R. R. Reed
(30) and Dr. W. J. Tang
(31) . For both types,
hybridizations were performed with the full-length cDNA. All of the
cDNA probes were labeled by random priming with
[
Hybridization of poly(A)
At mid-pregnancy,
clonidine potentiation of activated adenylyl cyclase was completely
abolished with G
The values are mean ± S.E. of four
independent determinations. Binding data were analyzed using an
iterative nonlinear least squares technique. The
K
Adenylyl cyclase activity was measured as described under
``Experimental Procedures'' in the presence of isoproterenol
0.1 mM, propranolol 10 µM, GTP 0.1 mM;
RM/1 antiserum was used at 1:50 dilution. Adenylyl cyclase activities
are expressed as total pmol of cAMP/mg of protein/10 min. Results are
presented as the mean ± S.E. of the number of determinations
indicated in parentheses. * indicates a significant difference between
membranes from mid-pregnancy and term ( p < 0.05; nonpaired
Student's t test).
We thank S. Cotecchia for helpful discussion and
critical reading of the manuscript. We thank D. E. Handy and C. S.
Flordellis for providing the cDNA for rat
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
- and
-adrenoreceptors (AR),
identified by Northern blotting in rat myometrium, showed a
differential expression during the course of pregnancy. Indeed, the
-AR transcript was present at mid-pregnancy, whereas
high levels of
-AR mRNA could be detected at term.
The role of these subtypes in modulating
-AR-stimulated adenylyl cyclase activity was
investigated on myometrial membranes from mid-pregnancy and term. At
nanomolar concentrations of clonidine (full
-AR
agonist) or oxymetazoline (partial
-AR agonist),
adenylyl cyclase activity was inhibited by up to 50 ± 7% at
mid-pregnancy or 75 ± 7% at term, whereas at micromolar
concentrations,
-AR agonists potentiate adenylyl
cyclase activity by 140-170% at mid-pregnancy. Both inhibitory
and stimulatory components of this biphasic response were blocked by
yohimbine, a selective
-AR antagonist. Preincubation
of myometrial membranes with G
and/or G
antisera eliminated
-AR mediated attenuation or
potentiation of isoproterenol-stimulated adenylyl cyclase, thus
indicating that both the inhibitory and stimulatory components are
mediated via G
and G
. In addition, type II
and IV adenylyl cyclases were identified by Northern blotting in the
pregnant rat myometrium. Altogether these data strongly suggest that
the
-AR at mid-pregnancy potentiates adenylyl cyclase
types II and IV through
released from G
and
G
proteins, whereas the
-AR expression
at term may be related to persistent inhibition.
-AR
(
)
induces myorelaxation via the G
proteins
coupled
-AR/cAMP cascade, whereas stimulation of the
-AR signaling pathway elicited contraction. We have
previously shown that progesterone, which culminates during
mid-pregnancy
(3) , enhances myometrial
-AR
responsiveness to catecholamines by controlling the rate of
transcription of the gene encoding the
-AR
(4) and by increasing the high affinity state,
R
, of this receptor
(5) . The existence of
multiple
-AR subtypes is supported by several lines of
evidence, the strongest of which are the cloning of three subtypes in
human and rat
(6, 7, 8, 9, 10, 11, 12, 13) ,
the development of specific drugs, and the characterization of several
cell lines that express a single subtype
(14, 15) .
These receptors, present in a variety of tissues or cell lines, were
both defined as receptors of high affinity for the agonists clonidine
and UK14304 and for the antagonists yohimbine and rauwolscine.
Recently, our pharmacological data strongly suggest that rat myometrium
could express the
- and the
-AR
subtypes
(16) . To date, in many systems examined,
-AR have been described as being coupled to G
with agonist occupancy resulting in a decrease of adenylyl
cyclase activity. However, the
-AR has also been
reported to stimulate adenylyl cyclase or cAMP accumulation in various
cell lines
(17, 18) or in stably transfected cells
(19, 20, 21, 22) . In addition, the
-AR may couple to other intracellular pathways
involving phospholipase A2, phospholipase C, and
Na
/H
exchange ion channels (for
review, see Refs. 23 and 24).
-AR expressed in
the rat myometrium during the course of pregnancy. Then, we examined
the question of whether activation of
-AR regulates
-AR-stimulated adenylyl cyclase activity. Finally, we
identified the G proteins involved in
-AR coupling and
the type(s) of adenylyl cyclase expressed in myometrium in order to
investigate by which mechanism
-AR may cross-regulate
the
-AR pathway.
Chemicals and Reagents
I-cAMP
assay system and [
H]rauwolscine (88 Ci/mmol) were
purchased from Amersham Corp.; [
-
P]dATP
(3000 Ci/mmol) and G protein antisera were obtained from DuPont NEN.
Pertussis toxin; isoproterenol; clonidine-, oxymetazoline-, and
yohimbine-hydrochloride; and all other reagents of the highest grade
commercially available were from Sigma. UK14304 tartrate was a gift
from Pfizer.
Animals
Sprague-Dawley rats were obtained from
Iffa-Credo (L'arbresle, France). The females were caged with
males overnight, and successful mating was determined by the presence
of spermatozoa in the vaginal smear (day 1 of pregnancy). In our
breeding colony, parturition occurs between 12 and 19 h on day 22 for
80% of rats.
Radioligand Binding Assays
The preparation of
membranes for the radioligand binding assays from pregnant myometrium
was as described previously
(16) . For competition experiments,
10 nM [H]rauwolscine (a concentration
near the K
value for that tissue) and
14-18 concentrations (1 pM to 1 mM) of
unlabeled competing drugs were added to duplicate tubes. Nonspecific
binding was determined in the presence of 10 µM
phentolamine. Binding data were analyzed using nonlinear least squares
curve-fitting program (Graph PAD Software) to obtain IC
values. IC
values were converted to
K
values by the Cheng and Prussoff
equation
(25) .
Adenylyl Cyclase Assays
Adenylyl cyclase activity
in myometrial cell membranes was measured as described previously
(5) . Briefly, 20-µl membrane fractions (2.5 mg of
protein/ml) were incubated in 100 µl of a medium containing 50
mM Tris (pH 7.4), 5 mM MgSO, 5
mM creatine phosphate, 12 units of creatine phosphokinase, 0.5
mM ATP, 1 mM isobutylmethylxanthine and 0.1% bovine
serum albumin, for 10 min at 30 °C. Reactions were stopped by
transferring the tubes to a boiling water bath for 3 min. After
centrifugation at 2500
g for 30 min at 4 °C, cAMP
accumulation in the supernatant was determined using the Amersham cAMP
radioimmunoassay system. When present in the assay, isoproterenol was
used at a concentration of 0.1 mM, which we have found to be
optimal for promoting
-AR stimulation of adenylyl
cyclase activity in the presence of 0.1 mM GTP
(5) .
Clonidine or oxymetazoline was used at a range of concentrations
between 0.1 nM and 1 mM. In some studies, membrane
suspensions were preincubated either with polyclonal antisera directed
against the carboxyl-terminal portion of G proteins (RM1,
anti-
; AS/7,
anti-
/
; EC/2,
anti-
/
; QL,
anti-
/
) for 2 h at 4 °C at a
dilution of 1:50, which is maximally effective to prevent functional
interaction between G protein and activated receptor or with 10
µg/ml of preactivated pertussis toxin for 30 min at 30 °C. We
have previously tested (data not shown) that, at this saturating
concentration, pertussis toxin causes full inactivation of myometrial
G
proteins.
RNA Preparation and Blotting
Total RNA was
extracted from myometrium and other tissues (brain, spleen, neonatal
and adult lung, kidney) by the cesium-trifluoroacetate gradient method
(26) as described previously
(4) . Poly(A) RNA were purified from total RNA using prepacked
oligo(dT)-cellulose columns (Pharmacia Biotech Inc.).
Poly(A)
RNA (10 µg) were then electrophoresed in a
1% agarose gel containing formaldehyde
(27) and transferred to
GeneScreen Plus membranes (DuPont NEN) by capillatory blotting.
cDNA Probes
Probes specific of each
-AR subtype were chosen from regions coding for the
putative third intracellular loop of the receptors since these
sequences are divergent between the different subtypes. The 333-base
pair KpnI- XmnI and 466-base pair
NaeI- StuI fragments were derived, respectively, from
the rat brain
and
cDNA (kindly
provided by Drs D. E. Handy and C. S. Flordellis) and were used as
subtype-specific probes
(28) . Transformation in Escherichia
coli, plasmid preparation, and cDNA purification were performed by
standard molecular biology methods
(29) . A fragment specific of
the
-subtype was amplified from rat genomic DNA using
polymerase chain reaction. The primers 5`-CGCAGCCACTGCAGAGGTCT-3`
(sense) and 5`-AGTCGCCCCACTAGTCCCCT-3` (antisense) were chosen and
provided amplification of a 279-base pair sequence from nucleic acids
981 to 1259
(10) . The polymerase chain reaction product was
subcloned into pCR
II vector (Invitrogen), and its sequence was
verified by the Sanger dideoxynucleotide termination method using T7
DNA polymerase (Pharmacia).
-
P]dATP to a specific activity averaging
10
dpm/µg. Unbound radioactivity was separated by gel
filtration using Sephadex G-50 DNA grade (Pharmacia).
Hybridization Procedures
The membranes were
prehybridized at 42 °C for 4 h in a buffer containing 45%
formamide, 4 SSC, 5
Denhart's solution, 100
mM NaH
PO
, pH 6.6, 0.5% SDS, and 75
µg/ml denatured salmon sperm DNA and then hybridized overnight at
42 °C in the same medium containing 10% dextran sulfate and
10
cpm/ml denatured cDNA probes. After hybridization,
membranes were washed with a final stringency of 0.1
SSC, 0.1%
SDS at 55 °C (adrenoreceptor probes) or 0.2
SSC, 0.2% SDS
at 55 °C (adenylyl cyclase probes) and exposed to Kodak X-Omat AR
films at -80 °C for 1-5 days. The intensities of the
bands were determined by scanning densitometry. Size estimates of the
RNA species were established by comparison with an RNA ladder (Life
Technologies, Inc.). Hybridization with an oligo(dT)
probe (Pharmacia) was used as an internal control to estimate the
amount of poly(A)
RNA loaded in each well and transfer
efficiency.
RNA with DNA
fragments specific for the three subtypes of the rat
-AR clearly demonstrates the presence and the
differential expression of the
- and
-AR genes in the rat myometrium during the course of
pregnancy. Fig. 1 A shows a high level of the 3.8-kb mRNA
of the
-subtype in rat myometrium. The
-AR-specific probe also hybridized with a 3.8-kb
transcript in rat tissues known to express this receptor subtype
(brain, spleen) and did not detect any signal in liver known to express
only the
-AR subtype (Fig. 1 A)
(28) . A weak hybridization to a band of 4.7 kb could also be
detected; the identity of this mRNA is still unknown
(32, 33) . A 4.2-kb message encoding the
-AR subtype was also found in myometrium
(Fig. 1 A) as in other tissues, which express this
subtype (for review, see Refs. 24 and 28). As expected from previous
data
(11) , the
-AR specific probe hybridized
to a single band of 3 kb in rat brain. No hybridization could be
detected in rat myometrium at any stage of pregnancy
(Fig. 1 A). As shown in Fig. 1 B, the
-AR transcript could be detected at all of the stages
of pregnancy. Quantification of the signal indicated that the
-AR mRNA is significantly higher (4-fold) at
mid-pregnancy (day 10 and day 12) than in the later stages, where it
remains constant. Conversely, the
-AR transcript
could not be detected until day 17 of pregnancy. The
-AR mRNA level then increases by 110% from day 17 to
term (Fig. 1 B).
Figure 1:
RNA blot analysis of
-AR subtype expression in rat myometrium. A,
identification of
-AR transcripts. Poly(A)
RNA (10 µg) isolated from myometrium ( M), brain
( B), spleen ( S), liver ( Li), kidney
( K), and neonatal lung ( nL) were hybridized to
P-labeled
-AR ( a),
-AR ( b), and
-AR
( c) cDNA probes, as described under ``Experimental
Procedures.'' Position of co-electrophoresed RNA size markers (in
kb) are shown. B, evolution of the steady-state level of
-AR ( a) and
-AR
( b) transcripts in rat myometrium during pregnancy.
Autoradiographic signals were quantified by densitometric scanning from
day 10 of pregnancy ( D10) to term ( T). The results
were expressed as the changes observed in the amount of
-AR subtype mRNA compared with values of day 21, as
arbitrary units. Values are mean ± S.E. of three to six
independent determinations. ★ indicates a statistically
significant difference from day 21 ( p < 0.05; nonpaired
Student's t test).
Since we have found that
-AR and
-AR subtypes were colocalized
in the same layer of the myometrium
(16, 34) , we then
explored the ability of selective
-AR agonists to
alter isoproterenol-stimulated adenylyl cyclase activity in myometrial
plasma membranes from mid-pregnancy or term. We first examined the
potency of clonidine, UK14304, or oxymetazoline to inhibit
[
H]rauwolscine binding to myometrial cell
membranes. As shown in , clonidine had a higher affinity
than UK14304 for the rat myometrial
-AR. On the other
hand, preincubation of myometrial membranes with the selective
-AR
antagonist propranolol or with the antiserum RM/1 raised against the
C-terminal decapeptide of
s-subunits abolished the
isoproterenol-mediated stimulation of adenylyl cyclase ().
Fig. 2
shows the dose-response curves for the effects of
clonidine on isoproterenol-stimulated adenylyl cyclase in myometrial
membranes; at mid-pregnancy, clonidine induced a biphasic response
exhibiting both inhibitory and stimulatory components with EC
values of 1.27 ± 0.01 and 1344 ± 72 nM,
respectively. Maximal inhibition (50 ± 7% versus 75
± 7% at term, p < 0.05) obtained at low
concentration (10 nM) of clonidine occurred when 20-25%
-AR are occupied as indicated by
[
H]rauwolscine competition data. At
mid-pregnancy, higher concentrations (1-100 µM) of
clonidine increased adenylyl cyclase activity to 140-170% over
that elicited by isoproterenol alone. Maximal stimulation of adenylyl
cyclase at 10 µM clonidine ( i.e. 1000-fold
greater agonist concentration) represents 85-90%
-AR occupancy. Inhibition and potentiation of
isoproterenol-stimulated adenylyl cyclase could be blocked by
coincubation with the
-AR-selective antagonist
yohimbine at a concentration that occupied 99% of
[
H]rauwolscine binding sites (Figs. 3 and 4).
These results demonstrated the implication of
-AR in
both inhibition and potentiation. Furthermore, at mid-pregnancy,
oxymetazoline also produced a biphasic response with 55 ± 1%
inhibition at 1 µM and 140 ± 7% potentiation at 1
mM. At the same range of concentrations, UK14304 elicited only
very minimal inhibition (31 ± 2%) and no potentiation of
isoproterenol-stimulated adenylyl cyclase activity. The lack of
efficiency of this imidazoline compound could be due to its lower
affinity for the rat myometrial
-AR (our present data
and Ref. 35). Thus, all other experiments have been conducted in the
presence of clonidine. By themselves, all three
-AR
agonists do not significantly activate basal adenylyl cyclase (data not
shown). Our results, obtained from in vivo studies, strongly
suggest that at mid-pregnancy activation of
-AR
subtype results in a biphasic response, whereas at term the highly
expressed
-AR transcript could be related to
persistent inhibition of adenylyl cyclase. These data resemble those
reported in cell lines stably transfected with
-
(19, 22) or
-AR
(18, 21) . In addition, agonist potencies in our
experiments are comparable with those from these other studies.
Figure 2:
Effects of clonidine on
isoproterenol-stimulated adenylyl cyclase activities in myometrial
plasma membranes. Adenylyl cyclase activity was measured as described
under ``Experimental Procedures.'' Data are expressed as
percent of isoproterenol-stimulated adenylyl cyclase activity (control
100%). Results are mean ± S.E. of four to seven independent
determinations. The dose response significance for inhibition and
potentiation was analyzed by one-way analysis of variance followed by
Ducan's multiple range test.
It
is well known that pertussis toxin ADP-ribosylates G proteins, which then remain in an inactive conformational state,
resulting in an ablation of their functional interaction with receptor
or effector enzyme. After treatment of myometrial membranes with this
toxin, the clonidine-induced inhibition of adenylyl cyclase activity
was completely abolished, indicating that coupling to a G
protein is involved. Furthermore, adenylyl cyclase activity was
2.5-3-fold higher than that measured in the presence of
isoproterenol alone (Fig. 3). In the absence of clonidine, when
membranes have been pretreated with pertussis toxin,
isoproterenol-stimulated adenylyl cyclase was also consistently
increased (110 ± 6 and 99 ± 13 pmol cAMP/mg of protein/10
min, respectively, at mid-pregnancy and term; the values are for three
experiments). To explain these data, it is conceivable to assume that
high concentrations (0.1 mM) of GTP, when present in the assay
system, were generally effective to activate the G
protein
signaling pathway, even in the absence of any inhibitory input. This
lead us to conclude that a tonic inhibition exists in the rat
myometrium during pregnancy that could be due to the high levels of
G
and G
proteins
(36, 37) . At
term, the level of ADP-ribosylated G
proteins was decreased
(38) , and this may account for the decline of adenylyl cyclase
stimulability (). These observations suggest a
physiological importance of these two competing pathways (G
and G
) in the coupling mechanisms that dictate
adenylyl cyclase responsiveness in the pregnant myometrium.
Figure 3:
Effects of yohimbine, pertussis toxin, and
G antisera on clonidine inhibition of isoproterenol-stimulated adenylyl
cyclase activity of myometrial membranes at mid-pregnancy ( A)
or term ( B). Adenylyl cyclase activity was measured as
described under ``Experimental Procedures'' in the presence
of isoproterenol (0.1 mM) and GTP (0.1 mM) plus
clonidine (100 nM). Yohimbine was used at the concentration of
10 µM. Membranes were preincubated with 10 µg/ml
pertussis toxin ( PTX) or with EC/2
(anti-/
) or/and AS/7
(anti-
/
) antiserum at a 1:50
dilution. Data are presented as net responses (total activity in the
presence of GTP 0.1 mM minus activity in the presence of GTP
alone). Results are expressed as the mean ± S.E. of the number
of independent determinations indicated in parenthesis.
★ indicates a statistically significant difference from
isoproterenol or from isoproterenol plus clonidine. ( p <
0.05; nonpaired Student's t test). NS, no
significant difference.
Preincubation of myometrial membranes with G antiserum
(AS/7) blocked the
-AR inhibitory pathway on both
mid-pregnancy and term, whereas G
antiserum (EC/2) was
only effective at term (Fig. 3). Thus, at term, the activated
-AR do interact functionally with both G
and G
proteins to inhibit the isoproterenol-mediated
effect on adenylyl cyclase activity. Indeed, Fig. 3 B demonstrates that uncoupling of G
alone from
-AR using AS/7 antiserum did not substantially reduced
adenylyl cyclase activity in response to clonidine. Similar responses
were observed when testing anti-G
alone, or anti-G
and anti-G
together (Fig. 3 B). These
data, compared with findings reported above after pertussis toxin
treatment of myometrial membranes, revealed that only a small fraction
of pertussis toxin-sensitive G
and G
proteins
do couple to
-AR to mediate clonidine inhibition of
isoproterenol-stimulated adenylyl cyclase at term.
or G
antiserum in isolation
or with both antisera together (Fig. 4). Our conclusion from this
findings is that the stimulatory component in the modulation of
adenylyl cyclase activity by
-AR, the only subtype
detected at mid-pregnancy, is due to dual coupling of G
and G
proteins to the
-AR. Since
potentiation by clonidine of adenylyl cyclase activity was unaffected
by QL antiserum, it is reasonable to consider that myometrial
-AR does not exert its effect via the phospholipase
C/Ca
/proteine kinase C signal transduction pathway
(39, 40, 41, 42) .
Figure 4:
Effects of yohimbine and G antisera on
clonidine potentiation of isoproterenol-stimulated adenylyl cyclase
activity of myometrial membranes at mid-pregnancy. Adenylyl cyclase
activity was measured as described under ``Experimental
Procedures'' in the presence of isoproterenol (0.1 mM)
and GTP (0.1 mM) plus clonidine (100 µM).
Yohimbine was used at the concentration of 10 µM.
Membranes were preincubated with AS/7
(anti-/
) or/and EC/2
(anti-
/
) or QL
(anti-
/
) antiserum or normal rabbit
antiserum at 1:50 dilution. Adenylyl cyclase activities are presented
as net responses (total activity in the presence of GTP (0.1
mM) plus adrenergic agents minus activity in the presence of
GTP alone). Results are expressed as the mean ± S.E. of the
number of determinations indicated in parenthesis. ★
indicates a statistically significant difference from isoproterenol or
from isoproterenol plus clonidine ( p < 0.05; nonpaired
Student's t test). NS, no significant
difference.
Recent studies
(31, 43, 44) have shown that type II and IV
adenylyl cyclases may be activated by released from G
proteins. Since, in our system, G
and G
mediate
-AR dependent potentiation of adenylyl
cyclase activity, we determined if one or both of these adenylyl
cyclases were expressed in the pregnant rat myometrium. Hybridization
of poly(A)
RNA from pregnant rat myometrium with the
type II adenylyl cyclase probe revealed a single band of 4.2 kb. As
expected, this message was also present (Fig. 5 A) in
brain and lung and very weakly expressed in kidney
(30) . High
levels of a 3.5-kb poly(A)
RNA were also detected with
the type IV probe (Fig. 5 B) in comparison with tissues
known to express adenylyl cyclase IV
(43) . Thus, a possible
molecular mechanism for the myometrial
-AR to
potentiate adenylyl cyclase activity could reside in the ability of
subunits released from
-AR-stimulated
G
proteins, in association with activated
, to stimulate adenylyl cyclase types II and IV. We
demonstrate above that dual coupling of
-AR to both
G
and G
is required to potentiate
isoproterenol-stimulated adenylyl cyclase. This lead us to suggest that
a high concentration of
is required for this stimulatory
effect, as predicted previously in membranes from Sf9 cells expressing
adenylyl cyclase II
(31) . Works are under progress in our
laboratory to confirm this interpretation by countering clonidine
potentiation of adenylyl cyclase activity in the presence of
subunit of retinal transducin (kindly provided by Dr T. Wieland) that
can bind and neutralize free
. At term, the absence of
potentiation of isoproterenol-stimulated adenylyl cyclase activity by
-AR, the highly expressed subtype at this stage of
pregnancy, could be related, in part, to the sharp decline of the
levels of G
proteins
(36, 37) and the
decrease of ADP-ribosylated G
(38) .
Figure 5:
RNA blot analysis of type II ( A)
and type IV ( B) adenylyl cyclase expression.
Poly(A) RNA (10 µg) isolated from brain
( B), myometrium ( M), lung ( L), and kidney
( K) were hybridized to a
P-labeled adenylyl
cyclase II or IV probe, as described under ``Experimental
Procedures''. Position of co-electrophoresed RNA size markers (in
kb) are shown.
In
conclusion, our data present the first detailed analysis of the
functionality and dual coupling of the -AR subtypes in
a physiological model. Interestingly, our data clearly prove the
observations made in vitro on transfected cell lines. These
findings could have important implications in the understanding of the
routing of signals that depends, for one tissue, on the molecular
diversity in receptors, G proteins, and effector systems and on
distinct pattern of their regulation. Further attempts are now
necessary to identify which factor(s) of pregnancy underly the
molecular mechanisms leading to the differential regulation of
myometrial adenylyl cyclase activity at mid-pregnancy versus term.
Table:
Affinities of -AR agonists for
inhibiting specific [
H]rauwolscine binding in
pregnant rat myometrium
value of
[
H]rauwolscine determined by Scatchard analysis
was 3.7 ± 0.7 nM.
Table:
Effects of GTP and isoproterenol on
adenylyl cyclase activity in myometrial plasma membranes of pregnant
rat
- and
-ARs, A. G. Gilman, R. R. Reed, and W. J. Tang for
the rat adenylyl cyclases II and IV cDNA. We also thank M. T. Robin for
expert assistance in Northern blotting analysis and illustration of the
manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.