Acute electrophysiological responses of bradykinin-stimulated human fibroblasts.
1. Acute responses to bradykinin in human dermal fibroblasts were studied at 20-24 degrees C using both the patch-clamp technique to monitor ion currents and Fura-2 fluorescence to monitor [Ca2+]i. 2. During subconfluent culture, human dermal fibroblasts can express a diversity of ion channels as described in the preceding paper. 3. When GTP (1 mM) was included in the pipette solution, two additional ion channel populations were transiently augmented in response to bradykinin stimulation. 4. The first is a component of outwardly rectifying current which reached maximal induction within 10-15 s after bradykinin addition (1 microM) and then decayed back to near baseline over 60 s. 5. Ion substitution experiments combined with tail current analysis indicate that the outward current is carried predominantly by K+. 6. Video imaging of single-cell Fura-2 fluorescence from both intact cells and patch-clamped cells showed temporal correlation of the K+ current modulation and the Ca2+ transients in response to bradykinin stimulation. 7. The calcium ionophore, ionomycin, caused both an increase in intracellular calcium and the augmentation of the outward K+ current. The amount of additional K+ current was correlated with [Ca2+]i levels and could be elicited even without the presence of GTP in the pipette. 8. Apamin, a blocker of Ca(2+)-activated K+ channels, inhibited (at 1 microM) the ionomycin-induced modulation of K+ current. 9. In addition, an inward current was transiently induced in response to bradykinin. This current was strictly dependent on the presence of GTP in the pipette solution. This current showed little voltage dependence, as evidenced by a linear current vs. voltage relation, and a reversal potential near but measurably more positive than 0 mV. 10. This current could be decoupled from the Ca2+ transient and be irreversibly induced by including GTP gamma S (100 microM) in the pipette solution. 11. Ion substitution experiments show that this is a non-specific cation channel. This current prefers monovalents but exhibits a small permeability to divalents. 12. GTP gamma S-induced single channels from isolated outside-out patches showed similar ion selectivity and voltage dependence. These channels are 32 pS in size with an estimated reversal potential of 17 mV.(ABSTRACT TRUNCATED AT 400 WORDS)
Estacion
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Department of Physiology and Biophysics, University of California, Irvine 92717.
D001920:Bradykinin / Q000494:pharmacology*; D015220:Calcium Channels / Q000187:drug effects; D004553:Electric Conductivity / Q000187:drug effects; D005347:Fibroblasts / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D015221:Potassium Channels / Q000187:drug effects; D012867:Skin / Q000166:cytology; D013268:Stimulation, Chemical; D013997:Time Factors
D015220:Calcium Channels; D015221:Potassium Channels; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D015759:Ionomycin; D006160:Guanosine Triphosphate; D001920:Bradykinin