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O24E�ect of carbachol on regulation of the mAChreceptor mRNA expression ADN insulin secretionin mouse pancreatic islets
SHI C-L, TAÈ LJEDAL I-B, MATTSSON M-OAddress of presenting author:Chun_Liang ShiDepartment of integrativa medical biologyUmea UniversitySwedenE-mail: [email protected]: 46-90-7865975; Fax: 46-90-7866696
Introduction. Mouse islets that are transplanted to the
kidney lose their responsiveness to the neurotransmit-
ters acetylcholine and noradrenaline (Shi & TaÈljedal
1995a,b, Shi et al, 1996). In an effort to understand how
islets may be affected by changes in microenvironment,
we studied the insulin secretion and the muscarinic
acetylcholine receptor messenger RNA (mAChR
mRNA) expression of islets kept in culture for 1±7 days.
Methods. Islets were isolated from BALB/c or ob/ob
mice. The effects of glucose and carbachol on islets
insulin secretion were studied by static incubation or
dynamic perifusion methods; insulin content extracted
by acid ethanol and ultrasonication; and mACh-
RmRNA level were investigated by the RT-PCR
method.
Results. Islet insulin content was not affected by the
glucose concentration in culture medium, nor by the
addition of carbachol into the culture medium (average
about 62 ng per lg islets dry weight). However, the
insulin secretion in response to glucose was markedly
decreased when islets were cultured in medium con-
taining 2 mmol/l glucose, in comparison to islets cul-
tured in medium containing 11.2 mmol/l (0.07 � 0.0
vs 2.56 � 0.4 ng per hr per 3 islets). The glucose-in-
duced insulin secretions was also decreased when islets
were cultured in medium containing cholinergic agaonist
carbachol in comparison with islets cultured without
carbachol (26.1 � 5.1 vs 55.9 � 3.5 ng per 25 min per
50 islets). Acetylcholine potentiated glucose-induced
insulin secretion in fresh islets and cultured islets. This
potentiating effect was diminished when islets were
cultured with carbachol (5.53 + 0.7 vs 1.99 � 0.1 ng per
60 min per 3 islets). When measured by RT-PCR
method, the expression of mAChR mRNA in pancreatic
islets was up-regulated after 3-days in vitro culture. The
up-regulation of mAChR mRNA was not affected by
adding carbachol in culture medium, neither by culture
islets in 2 mmol/l glucose.
Conclusions. 1. The mAChR mRNA was up-regulated
when pancreatic islets were isolated and kept in culture.
The mechanism is not known, but seems to be related
to the denervation states of islets, and not related to
glucose concentration in the culture medium. 2. Per-
sistent activation of mAChR by agonist carbachol
decreased glucose-induced insulin secretion and
diminished the potentiating efffect of acetylcholine.
REFERENCES
Shi C-L, & TaÈljedal I-B. 1995a. Transplantation 62, 1248±52.
Shi C-L, & TaÈljedal I-B. 1995b. Acta Diabetol 32, 116±20.
Shi C-L, Sehlin J, & TaÈljedal I-B. 1996. Eur J Endocrinol 135, 724±8.
O25Solute Permeabilities of Toad Skin under OutsideHypo-, Iso-, and Hyperosmotic Conditions
FRéSLEV J, LARSEN EHAddress of presenting author:August Krogh InstituteUniversitetsparken 13,3DK-2100 Kùbenhavn éAtt:Jeppe Frùslev, ZLE-mail: [email protected]: 3532 1643; Fax: 3532 1567
Introduction. Toad skin (Bufo bufo) exhibits net trans-
port of non-electrolytes in inward direction, drastically
enhanced by the addition of 200 mM urea to the out-
side bathing solution (Ussing & Johansen (1969)). As
water ¯ow was in outward direction the above trans-
port was denoted »anomalous solvent drag», but the
phenomenon is still unexplained. As a follow up on a
newly developed compartment model of leaky epithelia
(Larsen & Sùrensen (1999)), we have studied the effect
of different outside bathing solutions on unidirectional
tracer ¯uxes of sucrose, chloride, and sodium, with
conventional Ussing-chamber protocols under short-
curcuit conditions.
Methods. The composition of Ringer was (mM): Na+
(113.5), K+ (1.9), Ca++ (1.0), Cl) (114.8), and HCO3)
(2.4), pH� 8.2. Outside bath was either Ringer, Ringer
with 200 mM urea, or »freshwater» (5.7 mM Na+,
7.0 mM Cl) , others equal). When measuring sucrose
¯uxes, the solutions contained 2 mM sucrose. The
radio-isotope tracers were 14C-sucrose, 36Cl, and 22Na.
Results. We report a signi®cant net inward transport of
sucrose with hypo- (181 +/) 62 fmol/s/cm2
(mean +/) SEM), p < 0.02, n� 6), iso- (202 +/) 78
and 220 +/) 90 fmol/s/cm2 , p < 0.02, n� 6), and
hypertonic (2509 +/) 1021fmol/s/cm2 , p < 0.05,
n� 6) outside bathing solutions. Both in-, and out-
¯uxes increased �12 times after addition of urea to
outside bath (270 +/) 96 to 3101 +/) 1119 fmol/s/
cm2, and 51 +/) 9 to 592 +/) 120 fmol/s/cm2
A18 Ó 1999 Scandinavian Physiological Society
Acta Physiol Scand 1999, 167, A3±A28
