Regulation of L-alanine transport systems A and ASC by cyclic AMP and calcium in a reptilian duodenal model
Tomás Gómez,
Virtudes Medina,
Cristina M. Ramírez,
Rosa Dópido,
Antonio Lorenzo and
Mario Díaz*
Laboratorio de Fisiología Animal, Departamento de
Biología Animal, Universidad de La Laguna, 38206 Tenerife,
Spain
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Fig. 1. Effects of (A) forskolin (10 µmol l–1), (B)
theophylline (3 mmol l–1) and (C) dibutyryl cyclic AMP
(db-cAMP; 500 µmol l–1) on unidirectional and net
L-alanine fluxes across the duodenum of Gallotia galloti. Experiments
were carried out under short-circuit conditions in the presence of sodium. The
concentration of L-alanine was 1 mmol l–1 throughout the
experiments and was added to both serosal and mucosal bathing solutions 20 min
before the control values were obtained. Drugs were added in small volumes
(<50 µl) to both sides of the tissue immediately after the last control
values were obtained and were left for an additional 10 min before test
samples were taken (see Materials and methods). Results are means ±
S.E.M. for 16, 12 and 8 different duodenal segments, respectively.
Jms, mucosa-to-serosa flux; Jsm,
serosa-to-mucosa flux; Jnet, net flux.
*P<0.05; **P<0.01 compared with control.
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Fig. 2. Time-course for cyclic AMP production in response to (A) forskolin (10
µmol l–1) and (B) theophylline (3 mmol
l–1) in the duodenal mucosa of Gallotia galloti.
Forskolin and theophylline were added at time zero. Each point represents the
mean ± S.E.M. for six separate determinations. *P<0.05;
**P<0.01 compared with time 0.
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Fig. 3. Effects of (A) calcium ionophore A23187 and (B) extracellular calcium
replacement on duodenal unidirectional and net L-alanine fluxes. Experiments
were carried out under short-circuit conditions in the presence of sodium. The
concentration of L-alanine was 1 mmol l–1 throughout the
experiments and was added to both serosal and mucosal bathing solutions 20 min
before the control values were obtained. A23187 (0.5 µmol
l–1) was added to both sides of the tissue immediately after
the last control values were obtained. Calcium replacement was achieved by
replacing all the standard solution with a Ca2+-free solution
containing 0.5 mmol l–1 EGTA. Tissues were left for an
additional 10 min before test samples were taken (see Materials and methods).
Results are means ± S.E.M. for 16 and 10 duodenal segments,
respectively. Jms, mucosa-to-serosa flux;
Jsm, serosa-to-mucosa flux; Jnet, net
flux. **Statistically different from its corresponding control value at
P<0.01.
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Fig. 4. Effects of dibutyryl cyclic AMP (db-cAMP; 500 µmol l–1)
on isolated L-alanine transport systems across the duodenal mucosa of
Gallotia galloti. (A) Effects on Na+-independent
L-alanine transport. (B) Effects on system A activity as determined
by measuring the unidirectional and net MeAIB ( -methylamino-isobutiric
acid) fluxes (10 mmol l–1) in Na+-containing
solutions. (C) Effects on system ASC. System ASC transport activity was
obtained by determining L-alanine unidirectional and net fluxes in the
presence of a saturating concentration of MeAIB (20 mmol l–1)
in Na+-containing solutions. Effects of db-cAMP were measured 20
min after its addition to the bathing solutions. The concentration of
L-alanine in the bathing solutions was 1 mmol l–1. All
experiments were performed under short-circuit conditions. Results are means
± S.E.M. for 16 duodenal segments under each condition.
*P=0.05; **P=0.01 compared with control.
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Fig. 5. Effects of calcium ionophore A23187 (0.5 µmol l–1) on
isolated L-alanine transport systems across the duodenal mucosa of
Gallotia galloti. (A) Effects on Na+-independent
L-alanine transport. (B) Effects on system A activity as determined
by measuring the unidirectional and net MeAIB (-methylamino-isobutiric
acid) fluxes (10 mmol l–1) in Na+-containing
solutions. (C) Effects on system ASC. System ASC transport activity was
obtained by determining L-alanine unidirectional and net fluxes in the
presence of a saturating concentration of MeAIB (20 mmol l–1)
in Na+-containing solutions. A23187 effects were determined 20 min
after its addition to the bathing solutions. The concentration of L-alanine in
the bathing solutions was 1 mmol l–1. All experiments were
performed under short-circuit conditions. Results are means ± S.E.M.
for 16 duodenal segments under each condition. *P<0.05;
**P<0.01 compared with control.
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Fig. 6. Effects of (A) forskolin (Fsk; 10 µmol l–1) and (B)
calcium ionophore A23187 (0.5 µmol l–1) on
L-alanine-induced bioelectrical changes. Each bar represents the steady-state
value for the indicated manoeuvre within the experiment. The effects of drugs,
alone or in combination with inhibitors, were assessed by comparing the
magnitude of potential difference (PD) or short-circuit current
(Isc) changes upon consecutive additions of L-alanine
(L-ala). Values are expressed as a percentage of maximal change (% max
response) within each experiment. The insets show comparisons between
percentage PD and Isc changes in response to consecutive
L-alanine additions. Further details are provided in the text. Results are
means ± S.E.M. of 10 different duodenal segments. *P<0.05
compared with control;  P<0.05 compared with the
previous experimental condition. Ctrl, control; WO, washout; NS,
nonsignificant.
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© The Company of Biologists Ltd 2003
