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Cyclic nucleotide-dependent protein kinase inhibition by H-8: effects on ion transport
S M O'Grady1, H R DeJonge, A B Vaandrager
1Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
Abstract:
We explored the potential role of cyclic nucleotide-dependent protein phosphorylation in regulating ion transport across flounder intestinal mucosa by studying the effects of N-[2(methylamino)-ethyl]-s-isoquinolinesulfonamide (H-8), a selective inhibitor of cyclic nucleotide-dependent protein kinase in vitro. Addition of H-8 reversed the inhibitory effects of 8-bromoguanosine 3',5'-cyclic-monophosphate (8-BrcGMP), 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP), atriopeptin III (AP III), and vasoactive intestinal peptide (VIP) on the short-circuit current (Isc) and transepithelial potential difference (PD). Flux measurements established that these changes in Isc and PD directly reflected changes in Na and Cl absorption by the intestine. H-8 was unable, however, to reverse the inhibitory effects on Isc and PD of the Ca ionophore ionomycin and of substance P at dosages exceeding those needed to reverse the effects of AP III, VIP, and the cyclic nucleotides. We conclude that 1) H-8 (100 microM or less) does not exert toxic effects, 2) exogenously added cyclic nucleotide analogues inhibit ion transport through activation of cyclic nucleotide-dependent kinases resulting in protein phosphorylation, 3) activation of these kinases is an essential intermediate step in the inhibitory action of AP III and VIP on ion transport, and 4) the Ca ionophore ionomycin and substance P appear to inhibit ion transport by a mechanism that is independent of cyclic nucleotide-dependent protein phosphorylation.
Insights
Cyclic nucleotide-dependent protein phosphorylation regulates ion transport in flounder intestine. This study shows that specific inhibitors block signaling pathways for cyclic nucleotides, atriopeptin III, and vasoactive intestinal peptide, but not calcium or substance P.
Area of Science:
- Physiology
- Molecular Biology
- Biochemistry
Background:
- Ion transport across intestinal mucosa is crucial for nutrient absorption and homeostasis.
- Cyclic nucleotide-dependent protein phosphorylation is a key regulatory mechanism in various cellular processes.
- Understanding these pathways is vital for comprehending intestinal function and dysfunction.
Purpose of the Study:
- To investigate the role of cyclic nucleotide-dependent protein phosphorylation in regulating ion transport in flounder intestinal mucosa.
- To determine if N-[2(methylamino)-ethyl]-s-isoquinolinesulfonamide (H-8), a protein kinase inhibitor, affects ion transport modulated by cyclic nucleotides, atriopeptin III (AP III), and vasoactive intestinal peptide (VIP).
- To elucidate the signaling pathways involved in ion transport regulation by different signaling molecules.
Main Methods:
- In vitro studies using flounder intestinal mucosa.
- Application of H-8, a selective inhibitor of cyclic nucleotide-dependent protein kinase.
- Measurement of short-circuit current (Isc) and transepithelial potential difference (PD) to assess ion transport.
- Flux measurements to quantify Na and Cl absorption.
- Testing the effects of cyclic nucleotide analogues (8-BrcGMP, 8-BrcAMP), AP III, VIP, Ca ionophore ionomycin, and substance P.
Main Results:
- H-8 reversed the inhibitory effects of 8-BrcGMP, 8-BrcAMP, AP III, and VIP on Isc and PD, indicating involvement of cyclic nucleotide-dependent kinases.
- Flux measurements confirmed that changes in Isc and PD reflected alterations in Na and Cl absorption.
- H-8 did not reverse the inhibitory effects of ionomycin and substance P, suggesting an alternative signaling pathway.
- H-8 at concentrations up to 100 microM did not exhibit toxic effects on the intestinal mucosa.
Conclusions:
- Cyclic nucleotide-dependent protein phosphorylation, activated by cyclic nucleotide analogues, AP III, and VIP, plays a significant role in inhibiting ion transport in flounder intestinal mucosa.
- The inhibitory actions of AP III and VIP are mediated through the activation of cyclic nucleotide-dependent kinases.
- The Ca ionophore ionomycin and substance P inhibit ion transport via a mechanism independent of cyclic nucleotide-dependent protein phosphorylation.