Sodium and anion transport across the avian uterine (shell gland) epithelium
1 Cardiac Rhythm Management, Medtronic Corporation, 7000 Central Avenue NE,
Minneapolis, MN 55432, USA
2 Departments of Physiology and Animal Science, University of Minnesota, 495
Animal Science/Veterinary Medicine Building, 1988 Fitch Avenue, St Paul, MN
55108, USA
* Author for correspondence (e-mail: ograd001{at}umn.edu)
Accepted 24 November 2004
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: eggshell formation, ENaC, bicarbonate secretion, acetazolamide, carbonic anhydrase, chicken, Gallus domesticus
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Previous studies of Na+ transport in vivo have
demonstrated net absorption when the shell gland was perfused with plasma-like
saline solution (Eastin and Spaziani,
1978b). Addition of ouabain, an inhibitor of
Na+-K+ ATPase activity, inhibited basal Na+
transport. In vivo studies also demonstrated net
HCO3- secretion during shell formation that was
inhibited by acetazolamide (Eastin and Spaziani,
1978a
,b
).
This result suggested that HCO3- transport was dependent
on carbonic anhydrase activity and that the mechanism for
HCO3- uptake presumably involved CO2
diffusion across the plasma membrane. These findings also indicated that
HCO3- secretion was transcellular rather than
paracellular and involved transport pathways located within the apical
membrane of the shell gland epithelium. Measurements of Cl-
transport in vivo showed net absorption under conditions where the
shell gland was perfused with plasma-like saline solution. Cl-
absorption was inhibited when the gland was treated with ouabain or with
acetazolamide (Eastin and Spaziani,
1978a
,b
).
Thus Cl- transport also appeared to be dependent on carbonic
anhydrase activity and coupled to HCO3- secretion.
Earlier Ca2+ flux experiments demonstrated net Ca2+
secretion into the lumen of the shell gland that was dependent on the presence
of HCO3- in the bathing solutions (Pearson and Goldner,
1973,
1974
;
Pearson et al., 1977
).
Biochemical and histochemical studies suggested that a Ca2+-ATPase,
associated primarily with tubular gland epithelial cells, was involved in
active Ca2+ secretion across the epithelium
(Yamamoto et al., 1985
;
Coty, 1982
;
Gay and Schraer, 1971
;
Gay and Mueller, 1973
). In
addition, vitamin D-dependent calcium binding proteins were also localized to
tubular gland epithelial cells, using immunohistochemical localization
techniques (Lippielo and Wasserman,
1975
). These results supported the hypothesis that the glandular
epithelium was the site for Ca2+ secretion and that the mechanism
of secretion involved an electrogenic Ca2+-ATPase located in the
apical membrane of these cells. The basis for the HCO3-
dependency of Ca2+ secretion was not determined.
Egg production rates and eggshell thickness both decrease with age
(Bahr and Palmer, 1989;
Joyner et al., 1987
).
Associated with these changes are decreases in the levels of vitamin
1
,25(OH)2D3 and the mass of medullary bone in
older hens (Bahr and Palmer,
1989
). Vitamin 1
,25(OH)2D3 is
synthesized from cholecalciferol through hydroxylation by the liver and kidney
(Soares and Ottinger, 1988
).
The active metabolite (1
,25(OH)2D3) regulates
calcium metabolism by increasing intestinal Ca2+ absorption, and by
increasing the mobilization of Ca2+ from bone
(Bahr and Palmer, 1989
). Its
synthesis by the kidney is regulated by estrogen
(Bar and Hurwitz, 1987
;
Baksi and Kenny, 1977
). It is
interesting to note that 1
,25(OH)2D3
concentration decreases with age even though plasma levels of progesterone and
estradiol-17ß are similar in young and older hens
(Bar and Hurwitz, 1987
).
Moreover, plasma levels of ionized Ca2+ do not significantly change
in hens between 33 and 122 weeks of age
(Bahr and Palmer, 1989
). Thus,
the reasons for decreased eggshell thickness may involve changes in shell
gland function rather than availability of Ca2+ for secretion. Such
changes could involve a decrease in the ability of the epithelium to transport
Ca2+ directly or perhaps changes in the coupling of Ca2+
transport with HCO3- secretion across the
epithelium.
The objective of the present study was to investigate the Na+ and Cl- transport properties of the shell gland epithelium from the domestic chicken Gallus domesticus under in vitro conditions where transepithelial voltage and ion concentration gradients could be controlled. Transepithelial Na+ and Cl- flux measurements and ion substitution experiments were performed to determine the ionic basis of the basal short circuit current Isc. The effects of epithelial Na+ and Cl- channel blockers were tested to identify apical membrane conductance pathways for these ions. The effects of cAMP on ion transport was determined using a cell-permeant cAMP analog and the effects of age and molting on both basal Na+ and anion transport were examined. The results showed that electrogenic transepithelial Na+ absorption was dependent on amiloride-sensitive Na+ channels present in the apical membrane and that a significant portion of the basal Isc was dependent on Cl- and HCO3-. The data also indicated that rates of Na+ and anion transport were altered with age and during molting, which could potentially contribute to the decrease in shell thickness often associated with eggs from older birds.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Histology
Freshly isolated, intact shell gland mucosa was cut into 1 cm2
pieces and fixed in 10% buffered formalin prior to paraffin embedding. The
embedded tissue was cut into 35 µm sections and stained with
Hematoxalin and Eosin. Slides were examined using a compound microscope (Nikon
diaphot, Niles, IL, USA) and images acquired with a digital camera (Coolsnap,
Millersville, MD, USA) at 100x (Fig.
1A) and 400x (Fig.
1B).
|
Tissue preparation and measurement of electrical parameters
Domestic chickens Gallus domesticus L. were maintained at the
Animal Care Facility in the College of Veterinary Medicine at the University
of Minnesota under a 12 h:12 h light:dark cycle. Hens were pre-anesthetized by
an intramuscular injection of ketamine (10 mg kg-1) followed by
intravenous administration of Beuthanasia solution (4 ml kg-1)
containing 50 mg ml-1 pentobarbital. The shell gland was
subsequently removed and bathed in ice-cold avian saline solution containing
(in mmol l-1) 150 NaCl, 5 KCl, 1 CaCl2, 1
MgCl2, 25 NaHCO3, 1 NaH2PO4, pH
7.4. The serosal muscle layers were removed by blunt dissection. The mucosa
was mounted in Ussing chambers and bathed with avian saline solution on both
sides of the tissue. The solutions were gassed with 95% O2/5%
CO2 and maintained at 40°C (avian core temperature).
Transepithelial potential difference (3.0±0.22 mV, N=30),
tissue conductance (17.3±0.62 mS cm2, N=30) and
short circuit current (Isc; 71.7±5.1 µA
cm-2, N=30) were measured with the ground electrode placed
in the luminal solution, using voltage clamp circuitry from JWT Engineering
Corporation (Kansas City, KS, USA). The chamber area was 0.64 cm2.
For experiments that involved anion substitutions, Cl- was replaced
with methane sulfonate and HCO3- with Hepes buffer to
maintain a constant pH of 7.4. Tissue conductance was calculated from voltage
and current measurements obtained at various intervals throughout the
experiment. Glucose (10 mmol l-1) was added to the basolateral
solution to sustain the metabolic activity of the tissue, and mannitol (10
mmol l-1) was added to the apical solution to balance osmotic
pressure without stimulating Na+-dependent glucose transport
mechanisms that might exist within the apical membrane. In all experiments,
100 nmol l-1 tetrodotoxin and 10 µmol l-1
indomethacin were added to the basolateral solution to block any spontaneous
activity of submucosal nerves and to inhibit basal prostaglandin secretion
from the epithelium and stromal cells.
Transepithelial Na+ and Cl- flux measurements
Tissues were prepared as described above. All flux measurements were
performed on tissues under short circuit conditions. After allowing 30 min for
the tissues to stabilize, 5.5 x104 Bq
22Na+ and 1.1 x105 Bq
36Cl- were added to either the luminal or basolateral
side of the epithelium and allowed to equilibrate for 30 min. The first flux
period (30 minduration) served as a control to establish basal rates of
Na+ and Cl- transport across the epithelium.
Unidirectional fluxes were determined from measurements of isotope (0.1 ml)
recovered from the reservoir where isotope was initially added and from 1.0 ml
of saline solution from the opposite reservoir. Subsequently, the epithelium
was treated with 10 µmol l-1 amiloride for 5 min, and a second
30 min flux measurement was performed. 22Na+ was
measured using an LKB gamma counter and 36Cl- was
detected using a Beckman liquid scintillation counter. The net flux across the
epithelium was determined by subtracting the basolateral-to-apical
unidirectional flux from the apical-to-basolateral flux using paired tissues
from the same animal. Tissues were paired on the basis of transepithelial
conductance within 10%.
Statistics
Results are presented as the mean ± standard error
(S.E.M.). For statistical comparisons of flux data and
Isc responses between tissues obtained before and after
ion replacement or drug treatment (where the same tissue was used as control),
a paired t-test was used. An unpaired t-test was used for
comparisons between Isc responses from animals in
different age groups or during molting. The level of significance was set at
P<0.05.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The shell gland epithelium exhibited a basal Isc that ranged from 40100 µA depending on the stage of egg production. Addition of the Na+ channel blockers amiloride or benzamil to the apical bathing solution produced inhibition of the basal current by approximately 6070%. Inhibition of Isc was concentration dependent for both blockers with IC50 values and a rank order of potency consistent with inhibition of ENaC Na+ channels (Fig. 2). Interestingly, the magnitude of amiloride-sensitive Isc was nearly twofold greater in birds following completion of shell formation compared to hens where shell deposition was incomplete.
|
To confirm that the decrease in Isc produced by apical addition of amiloride was a consequence of inhibition of Na+ absorption, transepithelial Na+ and Cl- fluxes were measured (Fig. 3). These experiments were performed under short circuit conditions where the transepithelial voltage was clamped at 0 mV and identical saline solution was used on both sides of the tissue. Amiloride (10 µmol l-1) produced a significant decrease in the apical-to-basolateral unidirectional flux and in the net flux for Na+. However, treatment with amiloride did not completely block all of the net Na+ flux, suggesting that Na+ channel activity was not completely blocked at 10 µmol l-1 (consistent with the additional decrease in Isc observed with 100 µmol l-1 amiloride in Fig. 2C) or that electroneutral pathways for Na+ transport exist in the apical membrane. No significant effects of amiloride were observed on unidirectional or net Cl- fluxes.
|
Ion replacement experiments indicated that the basal, amiloride-insensitive Isc was both Cl- and HCO3- dependent (Fig. 4). In addition, treatment with the Cl- channel blocker, DPC, produced a concentration dependent decrease in Isc that was quantitatively similar to the Cl- and HCO3- dependent current (Fig. 4B). Moreover, transepithelial Cl- flux experiments showed a net Cl- flux, indicating Cl- secretion that was significantly different from zero under basal conditions. Basolateral addition of 8-cpt cAMP produced a rapid and sustained increase in Isc that was dependent on Cl- and HCO3- in the bathing solutions (Fig. 5). Addition of the acetazolamide (100 µmol l-1) to both apical and basolateral bathing solutions reduced most of the residual cAMP-stimulated Isc under Cl- free conditions, suggesting that HCO3- secretion was responsible for the residual current (Fig. 5B).
|
|
The effects of age and molting on basal, electrogenic Na+ and anion transport are shown in Fig. 6. The amiloride-sensitive Isc increased with age and during molting (Fig. 6A). In contrast, the anion-dependent basal current was significantly decreased in older birds (Fig. 6B). Interestingly, molting birds exhibited a basal Cl- dependent Isc similar to that observed for hens within the 5572 week age group, but the HCO3- dependency was significantly lower. This result is consistent with the interpretation that Cl- secretion uncouples from HCO3- transport in molting birds.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
An interesting observation from this study was the approximate twofold
increase in amiloride-sensitive Isc that occurred in birds
where shell formation was complete. One possible reason for this increase in
Na+ absorption may be to reduce fluid volume within the shell gland
lumen and to recover the Na+ and Cl- that were
transported across the epithelium during shell deposition. This interpretation
is consistent with earlier results from in situ perfused shell gland
experiments where net decreases in luminal Na+ and Cl-
concentrations were observed (Eastin and
Spaziani, 1978a). Moreover, previous studies of
Na+-K+ ATPase expression in chicken shell gland showed
that mRNA levels of the
1 subunit varied depending on the stage of
shell formation and exhibited different expression patterns within the surface
pseudostratified epithelium and the glandular epithelium
(Lavelin et al., 2001
). The
1 subunit mRNA levels were relatively low prior to entry of the egg
into the shell gland. Maximum increases in mRNA expression were subsequently
detected during the period of peak shell deposition and rapidly decreased once
shell formation was complete, 1 h prior to oviposition. Although changes in
1 subunit protein expression were not measured, it is reasonable to
assume that changes in mRNA levels preceded changes in functional activity of
the enzyme. Given this assumption, the increase in amiloride-sensitive
Isc observed in this study appeared to correlate with the
enhanced expression of the Na-K ATPase
1 subunit. Whether a similar
increase in Na+ channel expression also occurred is possible, but
remains to be established.
Anion substitution experiments revealed that the shell gland epithelium
exhibited a basal anion dependent Isc consistent with
anion secretion. Apical addition of the Cl- channel blocker DPC
inhibited the Cl- and HCO3- dependent
current, again suggesting net anion secretion under basal conditions. It is
worth noting in these experiments that tissues were pretreated with apical
amiloride to block basal Na+ channel activity and to ensure that
changes in Isc produced by DPC were not partially due to
effects on the driving force for Na+ uptake across the apical
membrane. Although DPC is a well established inhibitor of Cl-
channel activity, it is not highly selective, thus the molecular identity of
the DPC-sensitive conductance in the apical membrane could not be determined.
The suggestion that anion substitution and DPC inhibition of
Isc represented effects on basal anion secretion was
supported by measurements of transepithelial Cl- fluxes, which
demonstrated net Cl- secretion under basal conditions. Moreover,
stimulation with 8-cpt cAMP produced a sustained increase in
Isc that was significantly reduced if either
Cl- or HCO3- were replaced in the bathing
solutions. Attempts to measure transepithelial Cl- fluxes following
treatment with 8-cpt cAMP were confounded by increases in both unidirectional
Cl- fluxes (ranging between 2030%), making it difficult to
measure changes in the net flux accurately. Thus we were unable to confirm
cAMP stimulation of net Cl- secretion using isotopic flux
measurements. However, under symmetric saline solution conditions,
Isc responses to 8-cpt cAMP were stable in spite of
changes in paracellular permeability, thus ion substitution experiments could
be performed to investigate the anion dependence of the cAMP response. Based
on these findings, we speculate that the elevated Isc
resulting from cAMP stimulation was due to an increase in anion secretion.
Regulation of shell formation through the cAMP signaling cascade could
conceivably be mediated by various autocrine factors including prostaglandins
and adenosine or by certain hormones such as vasopressin and oxytocin
(Saito et al., 1987;
Olson et al., 1978
). However,
the specific signaling molecules involved in regulation of Cl- and
HCO3- transport by the shell gland epithelium have not
been identified.
The observation that a portion of the basal Isc and the
8-cpt cAMP stimulated Isc were both Cl- and
HCO3- dependent suggested that transport of these anions
may be coupled, as described for pancreatic duct cells that secrete
HCO3- (Steward et
al., 2005). In these cells, the apical membrane contains both cAMP
activated Cl- channels and
Cl-HCO3- exchangers. Cl-
channels provide a pathway for Cl- recycling across the apical
membrane to sustain HCO3- efflux mediated by
Cl-HCO3- exchange activity. In the
shell gland epithelium, we propose that a mechanism for Cl- uptake
across the basolateral membrane must also exist to allow for transcellular
Cl- secretion. Net Cl- secretion would presumably be
necessary to maintain a luminal [Cl-] sufficient to support
HCO3- transport by
Cl-HCO3- exchange and to compensate
for paracellular absorption of Cl- resulting from the
lumen-positive transepithelial potential. To test this hypothesis, DIDS, an
inhibitor of Cl-HCO3- exchange, was
added to the apical solution to see if it blocked the
HCO3- dependent component of the basal
Isc. We observed a decrease in Isc of
31±6.3 µA (N=5) that was abolished when tissues were bathed
on both sides with HCO3- free saline solution. Although
this result appears to be consistent with the suggested model above it is
important to note that DIDS also blocks certain types of Cl-
channels, thus more direct methods will be required to establish the mechanism
of HCO3- secretion in this epithelium.
Stimulation of Cl- secretion by 8-cpt cAMP was previously
reported in cultured mammalian endometrial epithelial cells
(Deachapunya and O'Grady,
1998). However, native porcine endometrial tissues mounted in
Ussing chambers and treated with 8-cpt cAMP or with PGF2
, produced a
sustained increase in amiloride-sensitive Na+ absorption
(Vetter and O'Grady, 1996
;
Vetter et al., 1997
). This
increase in Na+ transport was not associated with an increase in
apical Na+ conductance, but appeared to be the result of an
increase in driving force for apical Na+ uptake produced by
activation of basolateral K+ channels
(Vetter et al., 1997
).
Decreases in eggshell quality resulting from a reduction in shell thickness
is a well-known problem in egg production
(Bahr and Palmer, 1989;
Bennett, 1992
;
Hughes et al., 1986
; Joyner et
al., 1977; Poggenpoel, 1986
;
Soares et al., 1988
). In the
present study, significant decreases in basal anion transport were observed in
birds older than 55 weeks. Moreover, in molting birds there appeared to be
significant downregulation of basal HCO3- transport that
was associated with the overall regression of the reproductive tract. To date,
no direct evidence for age-dependent decreases in Ca2+ secretion by
the shell gland epithelium have been reported, but reductions in
HCO3- secretion have been shown to significantly
decrease calcite deposition. Limiting HCO3- secretion by
reducing plasma CO2, which can occur in heat-stressed birds, or by
treating hens with carbonic anhydrase inhibitors, typically leads to reduced
shell thickness (Hughes et al.,
1986
; Odom et al.,
1986
; Mashaly et al.,
2004
; Lavelin et al.,
2001
). Thus decreased rates of HCO3-
secretion may contribute to the problem of poor shell quality often observed
in older birds.
In summary, the results of this study support the following conclusions. First, the shell gland epithelium actively transports Na+ by an electrogenic mechanism that involves apical, amiloride-sensitive Na+ channels. We propose that this mechanism of Na+ transport is necessary for reclaiming Na+, Cl- and fluid from the shell gland lumen following completion of calcite deposition. Second, the shell gland epithelium exhibits basal anion secretion, which can be stimulated by signaling molecules that increase intracellular [cAMP]. We speculate that rates of HCO3- secretion are regulated by changes in intracellular [cAMP] during the process of shell formation and that this is necessary for delivery of adequate amounts of HCO3- required for shell deposition. Finally, we conclude that anion transport decreases in older hens and speculate that this may have important consequences on shell thickness.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bahr, J. M. and Palmer, S. S. (1989). The influence of aging on ovarian function. Poult. Sci. 2, 103-110.
Baksi, S. M. and Kenny, A. D. (1977). Vitamin D3 metabolism in immature Japanese quail: effect of ovarian hormones. Endocrinol. 10,1216 -1221.
Bar, A. and Hurwitz, S. (1987). Vitamin D metabolism and calbindin (calcium-binding protein) in aged laying hens. J. Nutr. 117,1775 -1779.[Medline]
Bennett, C. D. (1992). The influence of shell thickness on hatchability in commercial broiler breeder flocks. J. Appl. Poultry Res. 1,61 -65.
Chan, L. N., Wang, X. F., Tsang, L. L., So, S. C., Chung, Y. W., Liu, C. Q. and Chan, H. C. (2001). Inhibition of amiloride-sensitive Na(+) absorption by activation of CFTR in mouse endometrial epithelium. Pflug. Arch. 443,S132 -S136.[CrossRef][Medline]
Coty, W. A. (1982). The hen oviduct shell gland contains high levels of calcium-stimulated ATPase activity. Fed. Proc. 41,463 .
Deachapunya, C. and O'Grady, S. M. (1998).
Regulation of chloride secretion across porcine endometrial epithelial cells
by prostaglandin E2. J. Physiol.
508, 31-47.
Deachapunya, C., Palmer-Densmore, M. and O'Grady, S. M.
(1999). Insulin stimulates transepithelial sodium transport by
activation of a protein phosphatase that increases
Na+-K+ ATPase activity in endometrial epithelial cells.
J. Gen. Physiol. 114,561
-574.
Eastin, W. C. and Spaziani, E. (1978a). On the control of calcium secretion in the avian shell gland (uterus). Biol. Reprod. 19,493 -504.[Medline]
Eastin, W. C. and Spaziani, E. (1978b). On the mechanism of calcium secretion in the avian shell gland (uterus). Biol. Reprod. 19,505 -518.[Medline]
Gay, C. V. and Schraer, H. (1971). Autoradiographic localization of calcium in the mucosal cells of the avian oviduct. Cell Tissue Res. 7, 201-211.
Gay, C. V. and Mueller, W. J. (1973). Cellular localization of carbonic anhydrase in avian tissues by labeled inhibitor autoradiography. J. Histochem. Cytochem. 21,693 -702.[Medline]
Hughes, B. O., Gilbert, A. B. and Brown, M. F. (1986). Categorisation and causes of abnormal egg shells: relationship with stress. Br. Poult. Sci. 27,325 -337.[Medline]
Joyner, C. J., Peddie, M. J. and Taylor, T. G. (1987). The effect of age on egg production in the domestic hen. Gen. Comp. Endocrinol. 65,331 -336.[CrossRef][Medline]
Lavelin, I., Meiri, N., Genina, O., Alexiev, R. and Pines, M. (2001). Na+-K+-ATPase gene expression in the avian eggshell gland: distinct regulation in different cell types. Am. J. Physiol. 281,R1169 -R1176.
Lippielo, L. and Wasserman, R. H. (1975). Flourescent antibody localization of the vitamin D-dependent calcium-binding protein in the oviduct of the laying hen. J. Histochem. Cytochem. 23,111 -116.[Abstract]
Mashaly, M. M., Hendricks, G. L., Kalama, M. A., Gehad, A. E., Abbas, A. O. and Patterson P. H. (2004). Effect of heat stress on production parameters and immune responses of commercial laying hens. Poult. Sci. 83,889 -894.[Medline]
Matthews, C. J., McEwan, G. T., Redfern, C. P., Thomas, E. J.
and Hirst, B. H. (1998). Absorptive apical
amiloride-sensitive Na+ conductance in human endometrial
epithelium. J. Physiol.
513,443
-452.
Odom, T. W., Harrison, P. C. and Bottje, W. G. (1986). Effects of thermal-induced respiratory alkalosis on blood ionized calcium levels in the domestic hen. Poult. Sci. 65,570 -573.[Medline]
Olson, D. M., Bielleir, H. V. and Hertelendy, F. (1978). Shell gland responsiveness to prostaglandins F2 and E1 and to arginine vasotocin during the laying cycle of the domestic hen (Gallus domesticus). Gen. Comp. Endocrinol. 36,559 -565.[CrossRef][Medline]
Palmer-Densmore, M., Deachapunya, C., Kannan, M. and O'Grady, S.
M. (2002). UTP-dependent inhibition of Na+
absorption requires activation of PKC in endometrial epithelial cells.
J. Gen. Physiol. 120,897
-906.
Pearson, T. W. and Goldner, A. M. (1973).
Calcium transport across avian uterus. I. Effects of electrolyte substitution.
Am. J. Physiol. 225,1508
-1512.
Pearson, T. W. and Goldner, A. M. (1974).
Calcium transport across avian uterus. II. Effects of inhibitors and nitrogen.
Am. J. Physiol. 227,465
-468.
Pearson, T. W., Pryor, T. J. and Goldner, A. M. (1977). Calcium transport across avian uterus. III. Comparison of laying and nonlaying birds. Am. J. Physiol. 232,E437 -E443.[Medline]
Poggenpoel, D. G. (1986). Correlated response in shell and albumen quality with selection for increased egg production. Poult. Sci. 65,1633 -1641.
Saito, N., Sato, K. and Shimada, K. (1987). Prostaglandin levels in peripheral and follicular plasma, the isolated theca and granulosa layers of pre- and postovulatory follicles, and myometrium and mucosa of the shell gland (uterus) during a midsequence-oviposition of the hen (Gallus domesticus). Biol. Reprod. 36, 89-96.[Abstract]
Soares, J. H., Jr and Ottinger, M. A. (1988). Potential role of 1,25 dihydroxycholecalciferol in egg shell calcification. Poult. Sci. 67,1322 .
Steward, M. C., Ishiguro, H. and Case, R. M. (2005). Mechanisms of bicarbonate secretion in the pancreatic duct. Annu. Rev. Physiol. 67,101 -133.
Vetter, A. E. and O'Grady, S. M. (1996). Mechanisms of electrolyte transport across the endometrium. Regulation by PGF2 alpha and cAMP. Am. J. Physiol. 270,C663 -C672.[Medline]
Vetter, A. E., Deachapunya, C. and O'Grady, S. M. (1997). Na+ absorption across endometrial epithelial cells is stimulated by cAMP-dependent activation of an inwardly rectifying K+ channel. J. Memb. Biol. 160,119 -126.[CrossRef][Medline]
Yamamoto, T., Ozawa, H. and Nagai, H. (1985). Histochemical studies of Ca-ATPase, succinate and NAD+-dependent isocitrate dehydrogenases in the shell gland of laying Japanese quails: with special reference to calcium-transporting cells. Histochem. 83,221 -226.[CrossRef]