Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2001 Nov 6;98(23):13049-54.
doi: 10.1073/pnas.221381398. Epub 2001 Oct 16.

A uniform extracellular stimulus triggers distinct cAMP signals in different compartments of a simple cell

T C Rich  1 , K A Fagan, T E Tse, J Schaack, D M Cooper, J W Karpen
Affiliations

A uniform extracellular stimulus triggers distinct cAMP signals in different compartments of a simple cell

T C Rich et al. Proc Natl Acad Sci U S A. .

Erratum in

  • Proc Natl Acad Sci U S A 2001 Dec 4;98(25):14744

Abstract

cAMP, the classical second messenger, regulates many diverse cellular functions. The primary effector of cAMP signals, protein kinase A, differentially phosphorylates hundreds of cellular targets. Little is known, however, about the spatial and temporal nature of cAMP signals and their information content. Thus, it is largely unclear how cAMP, in response to different stimuli, orchestrates such a wide variety of cellular responses. Previously, we presented evidence that cAMP is produced in subcellular compartments near the plasma membrane, and that diffusion of cAMP from these compartments to the bulk cytosol is hindered. Here we report that a uniform extracellular stimulus initiates distinct cAMP signals within different cellular compartments. By using cyclic nucleotide-gated ion channels engineered as cAMP biosensors, we found that prostaglandin E(1) stimulation of human embryonic kidney cells caused a transient increase in cAMP concentration near the membrane. Interestingly, in the same time frame, the total cellular cAMP rose to a steady level. The decline in cAMP levels near the membrane was prevented by pretreatment with phosphodiesterase inhibitors. These data demonstrate that spatially and temporally distinct cAMP signals can coexist within simple cells.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Distinct cAMP signals measured in different subcellular compartments. (A) Membrane-localized cAMP signals were detected in cell populations by using Ca2+ influx through C460W/E583M channels. Ca2+ influx caused a decrease in fluorescence (ΔF/F0, plotted with inverted polarity). In the absence of PDE inhibitors (−IBMX), 10 μM PGE1 (arrow) triggered a rise and fall in Ca2+. The decay of the Ca2+ response was prevented by 5-min pretreatment with 100 μM IBMX. (B) Total cellular cAMP accumulation was determined in cell populations by monitoring the conversion of [3H]ATP to [3H]cAMP. PGE1 (10 μM) caused cellular cAMP to rise to a plateau in the absence of PDE inhibitors. A steady increase in cAMP was observed when cells were pretreated with 100 μM IBMX for 5 min.
Figure 2
Figure 2
PGE1 triggers sustained AC and PDE activity. (A and B) Local cAMP changes in response to AC stimulation, monitored by Ca2+ influx through C460W/E583M channels. PGE1 (A) or forskolin (B) were applied at the indicated concentrations. (C) Rises in cAMP (monitored by Ca2+ influx) caused by 100 nM PGE1 (t = 0) and subsequent addition of 100 μM IBMX (1, 2, 5, 10, 20, or 50 min, superimposed traces). The slopes of the IBMX-induced responses (0.0044 ± 0.0008 s−1) were similar to each other and to the slopes of the initial PGE1-induced responses (0.0038 ± 0.0003 s−1). IBMX and RO-20–1724 had no effect on channel activity in excised membrane patches (11). (D) Second additions of 100 nM PGE1 (2, 5, 10, 20, or 50 min, superimposed traces) after the initial addition of 100 nM PGE1 (t = 0) gave little or no response.
Figure 3
Figure 3
Single-cell measurements of local cAMP signals. (A and B) Upper: Rapid application of PGE1 triggered transient inward currents through CNG channels (−20 mV). Two different cells monitored in perforated patch configuration. Lower: the corresponding cAMP signals calibrated as described in Materials and Methods. (C) Rapid application of PGE1 triggered a transient inward current (whole-cell configuration, −20 mV). Subsequent application of PGE1 and IBMX triggered an inward current that rose to a plateau. This current was blocked by 10 mM MgCl2 (characteristic of CNG channels). Dashed lines indicate either zero cyclic nucleotide-induced current (the current in 10 mM MgCl2) or zero cAMP. No PGE1-induced currents were observed in cells not expressing CNG channels. Several additional controls were done to ensure that the PGE1-induced signal was a rise and fall in cAMP. PGE1 had no direct effect on channels in excised membrane patches, and when it was applied to cells it did not affect the cAMP sensitivity, the conductance, nor the number of active channels. In addition, treatment of CNG-channel-expressing cells with PGE1 triggered little or no release of Ca2+ from internal stores and no measurable increases in cGMP (Figs. 5 and 6, which are published as supporting information on the PNAS web site, www.pnas.org).
Figure 4
Figure 4
A quantitative description of the localized transient cAMP response and the total cellular cAMP accumulation. (Inset) Two-compartment model of the cell with a diffusional restriction between the membrane-localized microdomain (compartment 1) and the bulk cytosol (compartment 2). See text for details. (A) Rapid activation of AC and slower activation of PDE shape the transient signal in the microdomain. The slow flux of cAMP from the microdomain allows low levels to accumulate in the cytosol. Note that even in the small volume of the microdomain, the concentration of CNG channels would be low (≈40 nM) and would not be expected to buffer the measured cAMP signal. (B) Total cAMP levels (microdomain and cytosol) reach a plateau. Dashed lines indicate zero cAMP.
See this image and copyright information in PMC

Comment in

  • The many dimensions of cAMP signaling.
    Schwartz JH. Schwartz JH. Proc Natl Acad Sci U S A. 2001 Nov 20;98(24):13482-4. doi: 10.1073/pnas.251533998. Proc Natl Acad Sci U S A. 2001. PMID: 11717418 Free PMC article. Review. No abstract available.

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Sunahara R K, Dessauer C W, Gilman A G. Annu Rev Pharmacol Toxicol. 1996;36:461–480. - PubMed
    1. Beavo J A. Physiol Rev. 1995;75:725–748. - PubMed
    1. Walsh D A, Perkins J P, Krebs E G. J Biol Chem. 1968;243:3763–3765. - PubMed
    1. Gray P C, Scott J D, Catterall W A. Curr Opin Neurobiol. 1998;8:330–334. - PubMed
    1. Francis S, Corbin J D. Crit Rev Clin Lab Sci. 1999;36:275–328. - PubMed

Publication types

LinkOut - more resources