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cAMP activates an ATP-conductive pathway in cultured shark rectal gland cells
H F Cantiello1, G R Jackson, A G Prat
1Renal Unit, Massachusetts General Hospital East, and Department of Medicine, Harvard Medical School, Charlestown 02129, USA.
Abstract:
The molecular mechanisms associated with ATP transport and release into the extracellular milieu are largely unknown. To assess the presence of endogenous ATP-conductive pathway(s) in shark rectal gland (SRG) cells, patch-clamp techniques were applied to primary cultures of SRG cells. Whole cell currents were obtained with either intracellular tris(hydroxymethyl)aminomethane (Tris) or Mg2+ salts of ATP (200 mM nominal ATP) and 280 mM NaCl bathing solution. Basal currents showed a sizable ATP permeability for outward movement of MgATP. Adenosine 3',5'-cyclic monophosphate (cAMP) stimulation significantly increased the whole cell conductance (with either intracellular Tris-ATP or MgATP). Symmetrical whole cell ATP currents were also observed after cAMP activation, thus consistent with ATP as the main charge carrier. The cAMP-inducible ATP currents were insensitive to the Cl- channel blockers 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, diphenylamine-2-carboxylate, and anthracene-9-carboxylic acid but were readily blocked by nifedipine (400 microM) and glibenclamide (400 microM). The nature of the electrodiffusional ATP movement was further assessed by single-channel analysis of either MgATP or Tris-ATP currents in excised inside-out patches, both spontaneous and after activation with protein kinase A. Single-channel ATP currents were inhibited by either nifedipine or glibenclamide. Thus SRG cells express endogenous ATP-permeable pathways both before and after cAMP stimulation. Electrodiffusional ATP movement by SRG cells may play a significant role in the transport and delivery of cellular ATP to the extracellular milieu, which may help coordinate the dynamics of the epithelial secretory response in this cell model.
Insights
Shark rectal gland cells possess ATP-conductive pathways that facilitate adenosine triphosphate (ATP) release. Cyclic adenosine monophosphate (cAMP) stimulation enhances these ATP transport mechanisms, suggesting a role in epithelial secretion.
Area of Science:
- Cellular Physiology
- Molecular Biology
- Ion Transport
Background:
- Extracellular adenosine triphosphate (ATP) signaling is crucial in various physiological processes.
- The mechanisms of ATP transport and release in epithelial cells remain largely uncharacterized.
Purpose of the Study:
- To investigate the presence and characteristics of endogenous ATP-conductive pathways in shark rectal gland (SRG) cells.
- To determine the role of cyclic adenosine monophosphate (cAMP) in regulating ATP transport in SRG cells.
Main Methods:
- Patch-clamp electrophysiology (whole-cell and single-channel) was employed on primary cultured SRG cells.
- Cells were stimulated with cAMP and protein kinase A to assess ATP currents.
- Pharmacological agents, including Cl- channel blockers, nifedipine, and glibenclamide, were used to characterize the ATP pathways.
Main Results:
- SRG cells exhibit basal outward ATP permeability, particularly for MgATP.
- cAMP stimulation significantly increased whole-cell conductance and ATP currents, indicating ATP as the primary charge carrier.
- cAMP-inducible ATP currents were insensitive to Cl- channel blockers but were inhibited by nifedipine and glibenclamide.
Conclusions:
- SRG cells possess endogenous, nifedipine- and glibenclamide-sensitive ATP-permeable pathways.
- These pathways are upregulated by cAMP stimulation.
- Electrodiffusional ATP movement in SRG cells likely contributes to extracellular ATP delivery and epithelial secretory response coordination.