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A human intestinal epithelial cell line (Intestine 407) is known to retain receptors for intestinal secretagogues such as acetylcholine (ACh), histamine, serotonin (5-HT) and vasoactive intestinal peptide (VIP). The cells were also found to possess separate receptors for secretin and ATP, the stimulation of which elicited transient hyperpolarizations coupled to decreased membrane resistances. These responses were reversed in polarity at the K+ equilibrium potential. The hyperpolarizing responses to six agonists were reversibly inhibited by quinine or quinidine. By means of Ca2(+)-selective microelectrodes, increases in the cytosolic free Ca2+ concentration were observed in response to individual secretagogues. The time course of Ca2+ responses coincided with that of hyperpolarizing responses. The responses to ACh and 5-HT were abolished by a reduction in the extracellular Ca2+ concentration down to pCa 7 or by application of Co2+. Thus, in Intestine 407 cells, not only the intestinal secretagogues, which are believed to act via increased cytosolic Ca2+ (ACh, 5-HT and histamine), but also those which elevate cyclic AMP (VIP, secretin and ATP) induce increases in cytosolic Ca2+, thereby activating the K+ conductance. It is likely that the origin of increased cytosolic Ca2+ is mainly extracellular for ACh- and 5-HT-induced responses, whereas histamine, VIP, secretin and ATP mobilize Ca2+ from the internal compartment. [ABSTRACT FROM AUTHOR]

In giant fibroblastic L cells, penetration of a conventional microelectrode brought about marked decreases in the membrane potential and input resistance measured with a patch electrode under tight-seal whole-cell configuration, and repeated hyperpolarizations were often observed upon penetration. Therefore, the question arose whether such leakage artifact is a causal factor for generation of the membrane potential oscillation even in giant L cells. During whole-cell recordings, however, regular potential oscillations were observed in the cells that had not been impaled with a conventional microelectrode, as far as the Ca2+ buffer was not strong in the pipette solution. Oscillatory changes in the intracellular potential were detected by extracellular recordings with a tight-seal patch electrode in the cell-attached configuration. Thus, the potential oscillation occurs even in the absence of penetration-induced leakage or without rupture of the patch membrane. Withdrawal of a micropipette from one cell was often found to induce marked cell damage and elicit oscillatory hyperpolarizations in a neighboring cell with a certain time lag. The longer the distance between the injured and recorded cells, the greater was the time lag. Application of the cell lysate on the cell surface also gave rise to oscillatory hyperpolarizations. After repeated applications of the lysate, the membrane became unresponsive (desensitized), suggesting the involvement of receptors for the lysate factor. The lysates of different cell species (mouse lymphoma L5178Y cells or human epithelial Intestine 407 cells) produced similar effects. The effective component was heat stable and distinct from ATP. Lysate-induced hyperpolarizations were inhibited by deprivation of extracellular Ca2+ and by application of a Ca2+ channel blocker (nifedipine) or a K+ channel blocker (quinine) in the same manner as spontaneous oscillatory hyperpolarizations. It is concluded that the mouse fibroblast exhibits membrane potential oscillations, when the cell was activated, presumably via receptor systems, by some diffusible factors released from damaged cells. [ABSTRACT FROM AUTHOR]

The oscillation of membrane potential in fibroblastic L cells is known to result from periodic stimulation of Ca2+-activated K+ channels due to the oscillatory increase in the intracellular Ca2+ concentration. These repeated hyperpolarizations were inhibited by putative calmodulin antagonists, trifluoperazine (TFP), N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and promethazine (PMZ), and the concentrations required for half-maximal inhibition were 25, 30 and 300 microM, respectively. These doses were lower than those for reducing the membrane resistance due to nonspecific cell damages. Another calmodulin antagonist, chlorpromazine (CPZ), was also effective, but CPZ-sulfoxide was not. Intracellular pressure injections of calmodulin-interacting divalent cations, Ca2+, Sr2+, Mn2+ and Ni2+, elicited slow hyperpolarizations, whereas Mg2+ and Ba2+, which are known to be essentially inert for calmodulin, failed to evoke any responses. The injection of purified calmodulin also brought about a similar hyperpolarization. Quinine, an inhibitor of Ca2+-activated K+ channels, abolished both Ca2+- and calmodulin-induced hyperpolarizations. TFP prevented Ca2+-induced hyperpolarizations. The TFP effect was partially reversed by the calmodulin injection. It is concluded that calmodulin is involved in the operation of Ca2+-activated K+ channels in fibroblasts. [ABSTRACT FROM AUTHOR]

Using Ca2+- and K+-selective microelectrodes, the cytosolic free Ca2+ and K+ concentrations were measured in mouse fibroblastic L cells. When the extracellular Ca2+ concentration exceeded several micromoles, spontaneous oscillations of the intracellular free Ca2+ concentration were observed in the submicromolar ranges. During the Ca2+ oscillations, the membrane potential was found to oscillate concomitantly. The peak of cyclic increases in the free Ca2+ level coincided in time with the peak of periodic hyperpolarizations. Both oscillations were abolished by reducing the extracellular Ca2+ concentration down to 10(-7) M or by applying a Ca2+ channel blocker, nifedipine (50 microM). In the presence of 0.5 mM quinine, an inhibitor of Ca2+-activated K+ channel, sizable Ca2+ oscillations still persisted, while the potential oscillations were markedly suppressed. Oscillations of the intracellular K+ concentration between about 145 and 140 mM were often associated with the potential oscillations. The minimum phase of the K+ concentration was always 5 to 6 sec behind the peak hyperpolarization. Thus, it is concluded that the oscillation of membrane potential results from oscillatory increases in the intracellular Ca2+ level, which, in turn, periodically stimulate Ca2+-activated K+ channels. [ABSTRACT FROM AUTHOR]