Action potentials and whole cell sodium current were recorded in canine epicardial, midmyocardial, and endocardial myocytes in normal sodium at 37 degrees C. Tetrodotoxin (TTX) reduced the action potential duration of midmyocardial cells to a greater degree than either epicardial or endocardial cells. Whole cell recordings in potassium-free and very-low-chloride solutions revealed a slowly decaying current that was completely inhibited by 5 microM TTX or replacement of external and internal sodium with the impermeant cation N-methyl-D-glucamine. Late sodium current density at 0 mV was 47% greater in midmyocardial cells and averaged -0.532 +/- 0.058 pA/pF in endocardial, -0.463 +/- 0.068 pA/pF in epicardial, and -0.785 +/- 0.070 pA/pF in midmyocardial cells. Neither the frequency dependence of late sodium current nor its recovery from inactivation exhibited transmural differences. After a 4.5-s pulse to -30 mV, late sodium current recovered with a single time constant of 140 ms. We conclude that a larger late sodium conductance in midmyocardial cells will favor longer action potentials in these cells. More importantly, drugs that slow inactivation of sodium channels will produce a nonuniform response across the ventricular wall that is proarrhythmic.

Two microelectrodes have been used to measure membrane potentials simultaneously in pairs of mouse pancreatic islet cells. In the presence of glucose at concentrations between 5.6 and 22.2 mM, injection of current i into cell 1 caused a membrane potential change in this cell, V1, and, provided the second microelectrode was less than 35 micron away, in a second impaled cell 2, V2. This result establishes that there is electrical coupling between islet cells and suggests that the space constant of the coupling ratio within the islet tissue is of the order of a few beta-cell diameters. The current-membrane potential curves i-V1 and i-V2 are very similar. By exchange of the roles of the microelectrodes, no evidence of rectification of the current through the intercellular pathways was found. Removal of glucose caused a rapid decrease in the coupling ratio V2/V1. In steady-state conditions, the coupling ratio increases with the concentration of glucose in the range from 0 up to 22 mM. Values of the equivalent resistance of the junctional and nonjunctional membranes have been estimated and found to change with the concentration of glucose. Externally applied mitochondrial blockers induced a moderate increase in the junctional resistance possibly mediated by an increase in intracellular Ca2+.

The role of Ca 2؉ in stimulus-response coupling in nonexcitable cells is still not well understood. The Ca 2؉ responses of individual cells are extremely diverse, often displaying marked oscillations, and almost nothing is known about the specific features of these Ca 2؉ signals that are important for the functional response of a cell. Using the RBL-2H3 mucosal mast cell as a model, we have studied the temporal relationship between changes in intracellular Ca 2؉ and serotonin secretion at the single-cell level using simultaneous indo-1 photometry and constant potential amperometry. Secretion in response to antigen never occurs until intracellular Ca 2؉ is elevated, nor is it seen during the first few oscillations in Ca 2؉ . Exocytotic events tend to be clustered around the peaks of oscillations, but excellent secretion is also seen in cells with sustained elevations in Ca 2؉ . Ca 2؉ release from stores in the absence of influx fails to elicit secretion. If refilling and continued release of Ca 2؉ from stores is prevented with thapsigargin, Ca 2؉ influx can still trigger secretion, suggesting that store-associated microdomains of Ca 2؉ are not required for exocytosis. Our findings demonstrate the importance of an amplitude-encoded Ca 2؉ signal and Ca 2؉ influx for stimulus-secretion coupling in these nonexcitable cells.Although it is generally agreed that an increase in Ca 2ϩ is both necessary and sufficient for the initiation of secretion in most excitable cells, the role of Ca 2ϩ in nonexcitable cells is less clear (1). The individual Ca 2ϩ responses of nonexcitable cells are often extremely heterogeneous, and many models have been proposed for the generation of these complex and often oscillatory patterns (2). Both amplitude-encoded and frequency-encoded Ca 2ϩ signals have been proposed (3, 4), and the availability of both mechanisms would allow multiple signaling pathways to be activated by Ca 2ϩ in a single cell (5). Hepatocytes, with their repetitive oscillations of constant amplitude but variable frequency are prime candidates for frequencymodulated Ca 2ϩ signaling (6), and it has now been shown that mitochondrial NAD(P)H production in these cells is indeed regulated by the frequency of Ca 2ϩ oscillations (7,8). In contrast, it seems clear that the secretory response of single rat salivary acinar cells is tightly coupled to the amplitude of the Ca 2ϩ response (9), as is ciliary beating in tracheal epithelial cells (10). Furthermore, it has recently been shown that differential activation of transcription factors in B lymphocytes is achieved via non-oscillatory Ca 2ϩ signals of different amplitudes and durations (11).In most cases, however, it has not been easy to determine the specific features of the Ca 2ϩ signal that are important for a physiological response. This is because sensitive methods for detecting function at the single-cell level and with high temporal resolution are not readily available. Furthermore, it now seems clear that additional signals, such as the activation of protein kinase C (12) ...

The single-channel recording technique was employed to investigate the mechanism conferring ATP sensitivity to a metabolite-sensitive K channel in insulin-secreting cells. ATP stimulated channel activity in the 0-10/zM range, but depressed it at higher concentrations. In inside-out patches, addition of the cAMPdependent protein kinase inhibitor (PKI) reduced channel activity, suggesting that the stimulatory effect of ATP occurs via cAMP-dependent protein kinase-mediated phosphorylation. Raising ATP between 10 and 500/~M in the presence of exogenous PKI progressively reduced the channel activity; it is proposed that this inactivation results from a reduction in ldnase activity owing to an ATP-dependent binding of PKI or a protein with similar inhibitory properties to the kinase. A model describing the effects of ATP was developed, incorporating these two separate roles for the nucleotide. Assuming that the efficacy of ATP in controlling the channel activity depends upon the relative concentrations of inhibitor and catalytic subunit associated with the membrane, our model predicts that the channel sensitivity to ATP will vary when the ratio of these two modulators is altered. Based upon this, it is shown that the apparent discrepancy existing between the sensitivity of the channel to low ATP concentrations in the excised patch and the elevated intracellular level of ATP may be explained by postulating a change in the inhibitor/kinase ratio from 1:1 to 3:2 owing to the loss of protein kinase after patch excision. At a low concentration of ATP (10-20 #M), a nonhydrolyzable ATP analogue, AMP-PNP, enhanced the channel activity when present below 10 #M, whereas the analogue blocked the channel activity at higher concentrations. It is postulated that AMP-PNP inhibits the formation of the kinase-inhibitor complex in the former case, and prevents phosphate transfer in the latter. A similar mechanism would explain the interaction between ATP and ADP which is characterized by enhanced activity at low ADP concentrations and blocking at higher concentrations.

4. The frequency characteristics of the excess noise could be analysed as the sum of I/f and 1/f2 components. While the 1/f component remained unaffected by the external application of 20 mM-tetraethylammonium (TEA) and either 2 mM-Mn2+ or 2 mM-Co2+, the 1/f2 component was suppressed by both Mn2+ and Co2+.5. The corner frequency, fc of the 1/f2 component depended on membrane potential, which was adjusted by adjusting the [K+]o jump. These results support the idea that f, in these experiments is a measure of the channel relaxation.6. Measurements of the input resistance in the frequency range from 0 to 25 Hz were used to obtain a rough estimate of the size of the channel conductance as 5 x 10-12 f-i