Publisher: Karolinska Institutet, Department of Women's and Children's Health
Types: Doctoral thesis
Subjects: MEDICAL AND HEALTH SCIENCES, MEDICIN OCH HÄLSOVETENSKAP, Na, K-A TPase, PKA, PKC, dopamine, ouabain, cell attachment, Ca2+, oscillations, steroid, transcriptional regulation, IP3R.
Na,K-ATPase, an integral membrane protein expressed in each eukaryotic
cell, serves as the major determinant of intracellular ion composition.
In the current study we investigated novel aspects of Na,K-ATPase
function and regulation.
It is well established that Na,K-ATPase activity is regulated by
reversible phosphorylation. New findings in this study are: 1) the level
of intracellular Ca 2. concentration determines the functional effects of
PKA and PKC-mediated Na,K-ATPase phosphorylation. At low [Ca 2+]i ,
activation of PKA or PKC resulted in inhibition of Na,K-ATPase activity,
while at high (Ca 2+]i a stimulatory effect was observed. 2) Dynamic
regulation of Na,K-ATPase activity plays a functional role in striatal
neurons. Activation of D1 subtype of dopamine receptors resulted in
inhibition of Na,K-ATPase activity. An additional unexpected and
intriguing finding was colocalization of DI and D2 subtypes of dopamine
receptors in virtually all striatal neurons. Activation of D2 receptors
had no effect on Na,K-ATPase activity. D1 and D2 receptor activation had
opposite effects on TTX-sensitive Na+-channels.
A role for Na,K-ATPase in the regulation of cell adhesion was
demonstrated. Partial inhibition of Na,K-ATPase activity significantly
reduced cell attachment to fibronectin. Our results suggest that this
effect is mediated by perturbation of normal Ca 2+ signaling and a
reduction of focal adhesion kinase activity. These findings indicate the
importance of Na,KATPase during development and differentiation.
A role for Na,K-ATPase as a signal transducer involved in transcriptional
regulation was demonstrated. Ouabain, the enclogenous steroid ligand of
Na,K-ATPase, was found to act as a biological inducer of slow [Ca 2+]i
oscillations. This signal activated the transcription factor NF-kappaB.
The oscillations required Ca 2+ influx from extracellular space and
release from intracellular stores. The IP3R, located in the ER membrane,
was found to play a pivotal role in initiation and maintenance of Ca 2+
oscillations. By use of FRET and co-immunoprecipitation studies, we
demonstrated an interaction between Na,K-ATPase and IP3R. This
interaction was enhanced by ouabain. Our data indicate a novel mechanism
for IP3R activation and a novel role for Na,K-ATPase as a modulator of I
P3R-mediated Ca 2+ release.
In conclusion, this thesis demonstrates that Na,K-ATPase has a
multifunctional role. In addition to being the dynamic modulator of ion
transport, Na,K-ATPase also serves as a receptor and signal transducer
involved in the regulation of diverse cellular functions.
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