As adenosine 5'-triphosphate (ATP) released from astrocytes can modulate many neural signaling systems, the triggers and pathways for this ATP release are important. Here, the ability of mechanical strain to trigger ATP release through pannexin channels and the effects of sustained strain on pannexin expression were examined in rat optic nerve head astrocytes. Astrocytes released ATP when subjected to 5% of equibiaxial strain or to hypotonic swelling. Although astrocytes expressed mRNA for pannexins 1-3, connexin 43, and VNUT, pharmacological analysis suggested a predominant role for pannexins in mechanosensitive ATP release, with Rho kinase contribution. Astrocytes from panx1(-/-) mice had reduced baseline and stimulated levels of extracellular ATP, confirming the role for pannexins. Swelling astrocytes triggered a regulatory volume decrease that was inhibited by apyrase or probenecid. The swelling-induced rise in calcium was inhibited by P2X7 receptor antagonists A438079 and AZ10606120, in addition to apyrase and carbenoxolone. Extended stretch of astrocytes in vitro upregulated expression of panx1 and panx2 mRNA. A similar upregulation was observed in vivo in optic nerve head tissue from the Tg-MYOC(Y437H) mouse model of chronic glaucoma; genes for panx1, panx2, and panx3 were increased, whereas immunohistochemistry confirmed increased expression of pannexin 1 protein. In summary, astrocytes released ATP in response to mechanical strain, with pannexin 1 the predominant efflux pathway. Sustained strain upregulated pannexins in vitro and in vivo. Together, these findings provide a mechanism by which extracellular ATP remains elevated under chronic mechanical strain, as found in the optic nerve head of patients with glaucoma.
ATPase activity was measured in samples of freshly dissected rabbit ciliary epithelium. The epithelium was ruptured in distilled water, frozen briefly, and incubated at 37 degrees C in a buffer containing 100 mM NaCl and 32P-labeled adenosine triphosphate (ATP). The rate of ATP hydrolysis by the epithelium was linear for as long as 45 min. Ouabain (1 mM) reduced the ATP hydrolysis rate by approximately 50%. When the epithelium was preincubated for 10 min. in the presence of 1 mM dibutyryl cyclic adenosine monophosphate (cAMP), the ouabain-sensitive (Na,K-ATPase) activity was diminished; ouabain-insensitive ATPase activity was not reduced. Preincubation of the epithelium with forskolin with isobutylmethylxanthine also reduced ouabain-sensitive ATPase activity. These observations suggest that the ciliary epithelium may have a mechanism for short-term modulation of Na,K-ATPase activity by cAMP. Such a mechanism could be linked to the ability of cAMP-dependent protein kinase to reduce Na,K-ATPase activity in the tissue.
Previous studies show Src family kinase (SFK) activation is involved in a response that stimulates Na,K-ATPase. Here, we tested whether SFK activation is involved in the Na,K-ATPase response to endothelin-1 (ET-1). Intact porcine lenses were exposed to 100 nM ET-1 for 5-30 min. Then, the epithelium was removed and used for Na,K-ATPase activity measurement and Western blot analysis of SFK activation. Na,K-ATPase activity was reduced by ∼30% in lenses exposed to ET-1 for 15 min. The response was abolished by the SFK inhibitor PP2 or the ET receptor antagonist, PD145065. Activation of a ∼61 kDa SFK was evident from an increase in Y416 phosphorylation, which reached a maximum at 15 min ET-1 treatment, and a decrease in Y527 phosphorylation. PP2 prevented SFK activation. Since Fyn, Src, Hck, and Yes may contribute to the observed 61 kDa band, these SFKs were isolated by immunoprecipitation and analyzed. Based on Y416 phosphorylation, ET-1 appeared to activate Fyn, while Src and Hck were inhibited and Yes was unaltered. ET-1 requires SFK activation to cause Na,K-ATPase inhibition. ET-1 elicits a different pattern of SFK activation from that reported earlier for purinergic agonists that stimulate Na,K-ATPase activity and activate Src. In the ET-1 response Src is inhibited and Fyn is activated. The findings suggest SFK phosphorylation is involved in a regulatory mechanism for Na,K-ATPase. Knowing this may help us understand drug actions on Na,K-ATPase. Faulty regulation of Na,K-ATPase in the lens could contribute to cataract formation since an abnormal sodium content is associated with lens opacification.
The uptake of 45Ca by the rabbit lens reached equilibrium within 20 hr; the steady state lens/medium ratio was 0.08. This ratio is similar to that found for the lens/medium distribution of extracellular markers. Efflux of 45Ca from the lens was rapid and insensitive to iodoacetate. Mathematical analysis of 45Ca efflux curves revealed that calcium efflux from the lens could be described solely upon the basis of passive movement from the extracellular space. It is concluded that the exchangeable calcium in the normal rabbit lens is predominantly located in the extracellular space.
