Actin depolymerization in the cyclic AMP-stimulated toad bladder epithelial cell, determined by the DNAse method.

R M Hays; U Lindberg

doi:10.1016/0014-5793(91)80340-9

Home/Archive/PubMed

PubMed · 1901549

Actin depolymerization in the cyclic AMP-stimulated toad bladder epithelial cell, determined by the DNAse method.

R M Hays·U Lindberg

View original source ↗Export metadata ↓

Abstract

Previous studies with the rhodamine phalloidin binding assay have shown that antidiuretic hormone and 8-Br-cAMP rapidly depolymerize F-actin in toad bladder epithelial cells. We have extended these studies with the DNAse inhibition assay and have found that in isolated epithelial cell suspensions, G-actin increases from 37 to 56% of total actin following 8-br-cAMP stimulation. The G-actin concentration in the epithelial cell greatly exceeds its critical concentration, indicating the requirement for a G-actin sequestering protein or proteins in this system.

Explore related subjects

8-Bromo Cyclic Adenosine Monophosphate Actins Animals Bufo marinus Cells, Cultured Deoxyribonucleases Epithelium Urinary Bladder Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

R M Hays, U Lindberg. 1991-03-25. Actin depolymerization in the cyclic AMP-stimulated toad bladder epithelial cell, determined by the DNAse method.. https://doi.org/10.1016/0014-5793(91)80340-9

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed1992

Cyclic adenosine 3'5'-monophosphate potentiates excitatory amino acid and synaptic responses of rat spinal dorsal horn neurons.

Intracellular recordings were made from rat dorsal horn neurons in the in vitro slice preparation to study the actions of cyclic adenosine 3',5'-monophosphate (cyclic AMP). In the presence of TTX, bath application of the membrane permeable analogue of cyclic AMP, 8-Br cyclic AMP (25-100 microM) caused a small depolarization of the resting membrane potential accompanied by a variable change in membrane input resistance. In addition, 8-Br cyclic AMP caused a long-lasting increase in the spontaneous synaptic activity and the amplitude of presumed monosynaptic excitatory postsynaptic potentials evoked in the substantia gelatinosa neurons by orthodromic stimulation of a lumbar dorsal root. When the fast voltage-sensitive Na conductance was blocked by TTX, 8-Br cyclic AMP enhanced in a reversible manner, the depolarizing responses of a proportion of dorsal horn neurons to N-methyl-D-aspartic acid (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), quisqualic acid (QA) and kainic acid (KA). The effects of 8-Br cyclic AMP on the resting membrane potential and the NMDA response of dorsal horn neurons were mimicked by reducing phosphodiesterase activity with bath application of 3-isobutyl-1-methylxanthine, but not by cyclic AMP applied extracellularly. Moreover, we have found that intracellular application of a protein inhibitor of cyclic AMP-dependent protein kinase (PKI) into dorsal horn neurons prevents the 8-Br cyclic AMP-induced potentiation of the NMDA response of these cells. These results suggest that in the rat spinal dorsal horn the activation of the adenylate cyclase-cyclic AMP-dependent protein kinase system may be involved in the enhancement of the sensitivity of postsynaptic excitatory amino acid (NMDA, AMPA, KA) receptors and modulation of primary afferent neurotransmission, including nociception.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1992

DdPK3, which plays essential roles during Dictyostelium development, encodes the catalytic subunit of cAMP-dependent protein kinase.

We have previously reported the analysis of DdPK3, a developmentally regulated putative serine/threonine kinase that shares approximately 50% amino acid sequence identity with metazoan cAMP-dependent protein kinase A (PKA) and protein kinase C, within their catalytic domains. Cells in which the DdPK3 gene has been disrupted do not aggregate but they are able to induce aggregation-stage genes in response to cAMP pulses and the prestalk-specific ras gene DdrasD in response to high continuous levels of cAMP but will not induce prespore gene expression. In this report, we present conclusive evidence that DdPK3 encodes the catalytic subunit of the Dictyostelium PKA. DdPK3 null cells lack kinase activity that phosphorylates a PKA-specific substrate and is specifically inhibitable by recombinant cAMP-dependent protein kinase inhibitor. DdPK3 expressed in Escherichia coli has PKA activity that is inhibitable by protein kinase inhibitor. When Ddpk3 null cells are complemented with DdPK3 expressed from an actin promoter on an extrachromosomal vector (low copy number), PKA activity is restored and the cells proceed to the slug stage but will not culminate, suggesting that properly regulated PKA activity is essential for culmination. Moreover, overexpressing DdPK3 in wild-type cells on integrating vectors (high copy number) from either an actin or prespore-specific promoter results in accelerated development and the ability to form mature spores in monolayer culture in the presence of high cAMP, a developmental potential lacking in wild-type cells.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1992

Redundant regulation of urokinase plasminogen activator transcription by the two major isozymes of cAMP-dependent protein kinase.

The regulation of urokinase plasminogen activator (uPA) gene expression by the two major cAMP-dependent protein kinase isozymes was studied in SC115 mouse mammary carcinoma cells using the site-selective cAMP analog approach. SC115 cells expressed both type I and type II cAMP-dependent protein kinase holoenzyme (at a ratio of 2:3), and selective, partial activation of each holoenzyme could be demonstrated in vitro using appropriate combinations of cAMP analogs. When cells were exposed to the same analog combinations, uPA expression was upregulated 2- to 4-fold when either holoenzyme I or holoenzyme II was targeted. For comparison, a high concentration (1 mM) of 8-bromo-cAMP, an analog that does not discriminate between kinase isoforms, up-regulated uPA 10-fold. These findings suggest that there are two pathways of cAMP-dependent regulation of uPA, one mediated by holoenzyme I, the other by holoenzyme II, and that the end result of activation of each pathway is the same. Differences in the mechanism whereby each pathway regulates uPA were searched for but not found. Both pathways were shown to be dependent on catalytically active enzyme, to be potentiated by retinoic acid treatment, and to regulate uPA transcriptionally. The most likely interpretation of these findings is that uPA transcription is mediated solely by the action of the common catalytic subunit, regardless of whether it originated from holoenzyme I or holoenzyme II.

8-Bromo Cyclic Adenosine Monophosphate↗