Parathyroid hormone (PTH) increases cytosolic free Ca concentration ([ Ca2+]i) by mechanisms that depend on extracellular Ca in both cultured renal proximal tubules and isolated rabbit connecting tubules (CNTs). In CNTs 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) mimics this action, implicating cAMP as a second messenger, and part of the rise, due to increased luminal membrane Ca entry, is likely related to Ca absorption. In cultured proximal tubules the rise in [Ca2+]i, presumably mediated by increased Ca entry across the basolateral plasmalemma, activates gluconeogenesis and shortens microvilli. In the present study we examined cAMP-mediated Ca entry across the basolateral membranes of CNT cells, an effect potentially related to cell activation. Single CNTs were dissected from rabbit kidneys and loaded with fura-2. [Ca2+]i was measured by dual-wavelength excitation during perfusion of isolated segments in vitro. With 1.8 or 2.0 mM Ca in the lumen and the bath, suffusate 8-BrcAMP increased [Ca2+]i within minutes in a dose-dependent fashion. The increase persisted as long as 8-BrcAMP was present and reversed on its withdrawal. With 0.1 microM Ca in the lumen and the bath, 8-BrcAMP, but not ionomycin, failed to increase [Ca2+]i, implying that extracellular Ca is the major source. In tubules perfused with 2 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to eliminate luminal Ca, but suffused with 1.8 or 2.0 mM Ca, 8-BrcAMP increased [Ca2+]i (though less so than with Ca in the lumen), implying Ca entry across basolateral cell membranes. This rise in [Ca2+]i was attenuated markedly by the presence of 50 microM LaCl3 in the bath.(ABSTRACT TRUNCATED AT 250 WORDS)
The role of poly(ethylene glycol) brush architecture in complement activation on targeted microbubble surfaces
