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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online May 29, 2001 as doi:10.1096/fj.00-0749fje. |
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Secretory Physiology Section,
* Gene Transfer Section, Gene Therapy and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 20892, USA;
Neurobiotechnology Center, Ohio State University, Columbus, Ohio 43210, USA;
Department of Anesthesiology, University of California, Los Angeles, California 90095, USA
3Correspondence: Bldg. 10, Room 1N-113, National Institutes of Health, Bethesda MD 20892, USA. E-mail: ambudkar{at}yoda.nidcr.nih.gov
SPECIFIC AIMS
In vitro studies with excised salivary gland tissue or dispersed cell preparations have previously demonstrated that 1) sustained activation of the ion channels that drive fluid secretion requires a sustained elevation in [Ca2+]i, and 2) sustained elevation of [Ca2+]i depends primarily on Ca2+ influx. Thus, Ca2+ influx has been implicated in the mechanism of salivary fluid secretion. However, this suggestion has not been directly demonstrated since the Ca2+ influx pathway(s) in nonexcitable cells have not yet been identified. To examine the involvement of store-operated calcium entry (SOCE) in the regulation of fluid secretion from salivary gland, we have assessed the physiological consequence(s) of adenovirus-mediated expression of hTrp1 in vivo in rat submandibular glands (SMG).
PRINCIPAL FINDINGS
1. Adenovirus-mediated expression of hTrp1 in HSG cells enhances
store-operated calcium entry
A recombinant E1--adenovirus encoding
HA-tagged hTrp1 (AdCMV-hTrp1) was used to direct the expression of
HA-hTrp1 in the human submandibular gland cell line, HSG. A
dose-dependent increase in the hTrp1 protein was detected in
AdCMV-hTrp1-infected HSG cells. HA-hTrp1 was localized in the plasma
membrane region of infected HSG. The muscarinic agonist carbachol (CCh)
or the intracellular Ca2+ pump inhibitor
thapsigarin (Tg) were used to stimulate
[Ca2+]i mobilization. CCh
induced [Ca2+]i increases
in noninfected and AdCMV-hTrp1-infected cells in a
Ca2+-containing medium. An initial rapid increase
in [Ca2+]i due to
internal Ca2+ release was followed by a
relatively sustained elevation of
[Ca2+]i as a result of
Ca2+ influx. The initial
[Ca2+]i increases in the
two groups of cells were not significantly different. However,
sustained [Ca2+]i was
significantly higher in cells infected with 5 MOI AdCMV-hTrp1 than in
noninfected cells or those infected with 1 MOI virus. CCh-stimulated
increases in [Ca2+]i in
noninfected or AdCMV-Luc-infected (used as control virus) cells were
similar. The sustained
[Ca2+]i increase induced
by Tg was significantly higher in cells infected with 5 MOI AdCMV-hTrp1
than in noninfected cells or those infected with 1 MOI of virus.
Tg-stimulated [Ca2+]i
increase was not altered in cells infected with AdCMV-Luc. In
aggregate, these data show that AdCMV-Trp1 can be used to express
functional Trp1 protein. Further, the effects of Trp1 expression on
SOCE are not due to changes in the internal Ca2+
store, internal Ca2+ release mechanism(s), or the
virus per se.
2. AdCMV-hTrp1-mediated expression of hTrp1 in vivo in rat
submandibular glands leads to an enhancement of submandibular fluid
secretion
Trp1 expression in vivo in SMG was achieved by administering
AdCMV-hTrp1 to the glands by retrograde ductal instillation. Three days
after infection, SMG ducts were cannulated and saliva was collected
from SMG after parasympathetic stimulation with pilocarpine. The same
glands were excised and used to detect Trp1. Pilocarpine-stimulated
fluid secretion obtained from AdCMV-hTrp1-infected glands (Fig. 1A
, left panel) was consistently higher than
AdCMV-Luc-infected glands (Fig. 1A
, right panel). Saliva
flow from AdCMV-hTrp1-infected SMG glands was 
fivefold higher than
from glands infected with the control virus (P<0.02,
Student’s t test; unstimulated saliva flow was not
affected, Fig. 1B
). The HA-tagged hTrp1 was detected in all
SMG infected with AdCMV-Trp1 but not in those infected with the control
virus. Figure 1C
(left panel) shows HA-Trp1 in the same six
glands used in Fig. 1A
(right panel shows data with
AdCMV-Luc infected glands). Detection with anti-Trp1 antibody showed an
increase in the total levels of Trp1 in AdCMV-hTrp1-infected glands
(Fig. 1D
). The expressed HA-hTrp1 was localized in the
basolateral plasma membrane of SMG acinar cells (Fig. 1E
).
In ductal cells, which are considered to be relatively water
impermeable, the localization of HA-hTrp1 appeared to be less distinct.
Thus, the in vivo localization of HA-hTrp1 in SMG acinar cells is
consistent with the predicted site of SOCE in exocrine gland acinar
cells and localization of the endogenous Trp1 in these cells.
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3. AdCMV-hTrp1-mediated expression of HA-hTrp1 in SMG enhances SOCE
in acinar cells
[Ca2+]i mobilization
was examined in acinar cells isolated from SMG 3 days after infection
with either AdCMV-Trp1 or AdCMV-Luc. The data in Fig. 2A
, B
show that sustained
[Ca2+]i in acinar cells
stimulated with either CCh or Tg is significantly higher in cells
isolated from AdCMV-hTrp1-infected SMG than from AdCMV-Luc-infected
SMG. 1) CCh-stimulated initial elevation in
[Ca2+]i was not different
in the two groups of cells and 2)
[Ca2+]i increase induced
by either CCh or Tg in cells in Ca2+-free medium
was not different between the two groups of cells. These data
demonstrate that AdCMV-hTrp1-infected acinar cells have a higher level
of SOCE. This increase is not due to effects on CCh-stimulated
Ca2+ signaling events, the
Ca2+ store status, or the basal
Ca2+ permeability of SMG acinar
cells.
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4. HA-hTrp1 associates with IP3Rs in vivo in salivary
glands
HA-hTrp1 was coimmunoprecipitated with IP3R
type, 1, 2, or 3 from solubilized crude membranes prepared from
AdCMV-hTrp1-infected but not AdCMV-Luc-infected glands. These data
suggest that the exogenous hTrp1 associates with the endogenous
IP3Rs in vivo in rat SMG. Further studies are
required to determine whether this association is involved in
regulating SOCE.
CONCLUSIONS
We have demonstrated by in vivo expression of Trp1 in SMG and by direct in situ measurements of agonist stimulated fluid secretion that SOCE is involved in the regulation of fluid secretion from SMG. Studies with isolated cell preparations from salivary glands have established that a Ca2+ influx pathway is activated shortly after agonist stimulation of cells. This Ca2+ influx determines the sustained elevation of [Ca2+]i in stimulated cells, which regulates several key ion channels that drive fluid secretion. Agonist-stimulated Ca2+ influx in salivary acinar cells, the site of fluid secretion, is mediated primarily via SOCE. However, the role of SOCE in agonist-stimulated fluid secretion has not yet been directly demonstrated in any exocrine gland mainly due to lack of knowledge regarding the molecular component(s) of the SOCE channel (SOCC).
Salivary glands provide an excellent system for gene transfer and transgene expression. Furthermore, studies using such approaches suggest that gene transfer can be a powerful tool to probe questions related to salivary gland physiology and pathophysiology. In the present study, we have used this approach to provide direct evidence for the suggestion, made more than three decades ago, that extracellular Ca2+ (and by inference Ca2+ influx) is involved in salivary gland fluid secretion. We have used an adenovirus encoding hTrp1 to direct expression of Trp1 in vivo in rat submandibular glands. We have recently proposed that Trp 1 is a possible candidate for the SOCC in salivary gland cells. Further, Trp1 is endogenously present in rat submandibular gland acinar cells. Here we have examined the physiological consequence of Trp1 expression in vivo in SMG.
Our data show that adenovirus-directed in vivo expression of hTrp1 enhances SOCE in acinar cells isolated from AdCMV-hTrp1-infected SMG. Furthermore, there is a dramatic increase in agonist-stimulated fluid secretion from glands infected with AdCMV-hTrp1 compared with those infected with AdCMV-Luc. The exogenous HA-tagged hTrp1 is expressed in the basolateral plasma membrane region of SMG acinar cells, the proposed site of SOCE in these cells. We conclude that hTrp1 expression in vivo in the basolateral plasma membrane of rat SMG acinar cells induces an increase in saliva flow from SMG by increasing pilocarpine-stimulated SOCE in the acinar cells. Based on these data, it is reasonable to suggest that the endogenous Trp1 protein in these acinar cells will also be activated under these conditions and thus have a role in the regulation of fluid secretion.
In summary, we have demonstrated a correlation between the level of Trp1 and agonist-stimulated saliva flow in rat SMG. Our data suggest that pilocarpine stimulation of SMG leads to the activation of the exogenous Trp1, and likely the endogenous Trp1, in acinar cells. The data indicate that SOCE is activated in SMG acinar cells after muscarinic receptor stimulation. Thus, we have directly demonstrated for the first time in vivo in rat SMG a causal relationship between SOCE, Trp1, and fluid secretion.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0749fje ; to cite this
article, use FASEB J. (May 29, 2001) 10.1096/fj.00-0749fje 
2 These investigators contributed equally to this work.
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