Larger late sodium conductance in M cells contributes to electrical heterogeneity in canine ventricle.

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.

Ito1 dictates behavior of ICl(Ca) during early repolarization of canine ventricle.

The contributions of the 4-aminopyridine (4-AP)-sensitive transient outward potassium conductance (Ito1) and the calcium-activated chloride conductance (ICl(Ca)] to cardiac action potentials were investigated in canine ventricular myocytes. Action potentials or currents were recorded at 37 degrees C using standard whole cell or amphotericin B perforated-patch-clamp techniques. Inhibition of Ito1 by 1 mM 4-AP prolonged phase 1 repolarization, elevated the action potential notch, and depressed the plateau. Action potential voltage clamp revealed that 4-AP blocked a rapidly decaying outward current during phase 1 without affecting plateau or diastolic currents. These results suggested that depression of the plateau was not a direct result of Ito1 inhibition but followed from delayed phase 1 repolarization. Calcium current (ICa) at the peak of the action potential dome was reduced 60 +/- 4% when the rate of phase 1 repolarization was reduced. ICl(Ca) measured by action potential clamp reversed over the course of the action potential. Chloride fluxes associated with outward and inward components of the 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid-sensitive current were +130 +/- 17 and -184 +/- 20 (pA.ms)/pF, respectively. The effects of selective inhibition of ICl(Ca) on the action potential were dependent on the rate of early repolarization and the prominence of the notch. Inhibition of ICl(Ca) elevated the plateau and slightly abbreviated action potential duration when the notch was prominent. When repolarization was prolonged and the notch was shallow, inhibition of ICl(Ca) elevated the notch and the plateau and abbreviated duration. We have shown that Ito1 and ICl(Ca) contribute to canine ventricular action potentials. The extent of overlap between Ito1 and ICl(Ca) during the action potential is largely determined by the amplitude of Ito1 and the depth of the notch. Regional differences in the density of Ito1, or interventions that moderate phase 1 repolarization by reducing this current, will have considerable effect on the time course of ICa and calcium-dependent conductances.

Correlating Ca2+ responses and secretion in individual RBL-2H3 mucosal mast cells.

The role of Ca2+ in stimulus-response coupling in nonexcitable cells is still not well understood. The Ca2+ responses of individual cells are extremely diverse, often displaying marked oscillations, and almost nothing is known about the specific features of these Ca2+ 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 Ca2+ 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 Ca2+ is elevated, nor is it seen during the first few oscillations in Ca2+. Exocytotic events tend to be clustered around the peaks of oscillations, but excellent secretion is also seen in cells with sustained elevations in Ca2+. Ca2+ release from stores in the absence of influx fails to elicit secretion. If refilling and continued release of Ca2+ from stores is prevented with thapsigargin, Ca2+ influx can still trigger secretion, suggesting that store-associated microdomains of Ca2+ are not required for exocytosis. Our findings demonstrate the importance of an amplitude-encoded Ca2+ signal and Ca2+ influx for stimulus-secretion coupling in these nonexcitable cells.

Characterization of the G protein coupling of SRIF and beta-adrenergic receptors to the maxi KCa channel in insulin-secreting cells.

Modulation of the Ca- and voltage-dependent K channel--KCa--by receptors coupled to the G proteins G(i)/G(o) and Gs has been studied in insulin-secreting cells using the patch clamp technique. In excised outside-out patches somatostatin (somatotropin-releasing inhibitory factor; SRIF) caused concentration-dependent inhibition of the KCa channel, an effect that was prevented by pertussis toxin (PTX). In inside-out patches, exogenous alpha subunits of either G(i)- or G(o)-type G proteins also inhibited the KCa channel (IC50 5.9 and 5.7 pM, respectively). These data indicate that SRIF suppresses KCa channel activity via a membrane-delimited pathway that involves the alpha subunits of PTX-sensitive G proteins G(i) and/or G(o). In outside-out patches, activation of Gs either by beta-agonists or with cholera toxin (CTX) increased KCa channel activity, consistent with a membrane-delimited stimulatory pathway linking the beta-adrenergic receptor to the KCa channel via Gs. In outside-out patches, channel inhibition by SRIF suppressed the stimulatory effect of beta-agonists but not that of CTX, while in inside-out patches CTX reversed channel inhibition induced by exogenous alpha i or alpha o. Taken together these data suggest that KCa channel activity is enhanced by activation of Gs and blocked by activated G(i) and/or G(o). Further, KCa channel stimulation by activated Gs may be "direct," while inhibition by G(i)/G alpha may involve deactivation of Gs. In inside-out patches KCa channel activity was reduced by an activator of protein kinase C (PKC) and enhanced by inhibitors of PKC, indicating that PKC also acts to inhibit the KCa channel via a membrane delimited pathway. In outside-out patches, chelerythrine, a membrane permeant inhibitor of PKC prevented the inhibitory effect of SRIF, and in inside-out patches PKC inhibitors prevented the inhibitory effect of exogenous alpha i or alpha o. These data indicate that PKC facilitates the inhibitory effect of the PTX-sensitive G proteins which are activated by coupling to SRIF receptors. To account for these results a mechanism is proposed whereby PKC may be involved in G(i)/G(o)-induced deactivation of Gs.

Characterization of the G protein coupling of a somatostatin receptor to the K+ATP channel in insulin-secreting mammalian HIT and RIN cell lines.

1. The G protein-mediated coupling of a somatostatin (somatotropin-releasing inhibitory factor; SRIF) receptor to the ATP-dependent K+ channel (K+ATP channel) has been studied in insulin-secreting cells using the patch clamp technique. 2. In excised outside-out patches, the concentration-dependent stimulation of the K+ATP channel by SRIF was biphasic. Stimulation reached a maximum at 15 nM (EC50 = 5.5 nM), then decayed to a minimum at 50 nM and returned to maximum stimulation at 500 nM. 3. In cell-attached patches, bath-applied SRIF caused K+ATP channel stimulation in most experiments. In a few cases, however, SRIF suppressed channel activity, a response that was reversed by addition of dibutyryl cyclic AMP (DBcAMP). Channel stimulation by SRIF or by DBcAMP did not occur in the presence of glucose. 4. In excised inside-out patches, the alpha-subunits of Gi or G(o)-type G proteins stimulated the K+ATP channel (EC50 = 29 and 42 pM, respectively). The K+ATP channel stimulation by alpha i- or alpha o-subunits had no effect on the concentration-dependent inhibition by ATP. 5. In excised inside-out patches, K+ATP channel activity was reduced by inhibitors of protein kinase C (PKC) and stimulated by a PKC activator. The stimulatory effect of PKC was unaffected by the presence of pertussis toxin, but stimulation by exogenous alpha-subunits of the G protein Gi or G(o) was prevented by PKC inhibitors. 6. From these data we deduce that SRIF can affect K+ATP channel activity directly via a membrane-delimited pathway or indirectly via a pathway requiring diffusible messengers. In the former case, alpha i/alpha o may either enhance PLC activity, stimulating PKC and thus inducing K+ATP channel phosphorylation with consequent increase of activity, or channel phosphorylation by PKC may facilitate a direct stimulation of the channel by alpha i/alpha o. In the latter case, an alpha i/alpha o-induced fall in cAMP contributes to reduced PKA-mediated phosphorylation and suppression of channel activity.

Mastoparan increases the intracellular free calcium concentration in two insulin-secreting cell lines by inhibition of ATP-sensitive potassium channels.

The mechanisms underlying mastoparan-induced elevation of the intracellular free calcium concentration ([Ca2+]i) were investigated in the insulin-secreting cell lines RINm5F and HIT. In both cell types, micromolar concentrations of mastoparan induced a prompt increase of [Ca2+]i, measured as an increase in fura-2 fluorescence. This response was dependent on extracellular calcium entry and was suppressed by organic calcium channel blockers; the increase of [Ca2+]i caused by high glucose concentrations or tolbutamide was not enhanced by mastoparan. These data indicate the involvement of voltage-dependent calcium channels and suggest that depolarization, rather than a direct effect on the channels, mediates the response to mastoparan. This proposition was supported by the observation that whole-cell calcium currents measured using the nystatin-permeabilized patch technique were not affected by mastoparan. Mastoparan-induced depolarization was observed using the potentiometric indicator bis-oxonol, and it was shown not to be additive with the depolarization induced by high glucose concentrations or tolbutamide. The mechanism underlying mastoparan-induced depolarization was identified in single-channel patch-clamp experiments, where it was shown that mastoparan caused closure of ATP-sensitive potassium channels [K(ATP) channels] in cell-attached and excised membrane patches. Responsiveness to mastoparan in excised patches demonstrated the membrane-delimited character of K(ATP) channel inhibition. The observation that the response persisted in the absence of exogenous GTP and in the presence of 250 microM GDP or guanosine-5'-O-(2-thio)diphosphate suggested that this effect is not mediated via enhancement of G protein activity. Partial suppression of channel activity by mastoparan did not prevent the action of tolbutamide, which fully suppressed the remaining activity in excised patches. In summary, the increase of [Ca2+]i in the insulin-secreting tumor cell lines RINm5F and HIT in response to mastoparan is mediated via G protein-independent suppression of K(ATP) channel activity, cell depolarization, and activation of voltage-dependent calcium channels.

Pancreatic beta-cell glucose hypersensitivity in hyperglycemic, athymic "nude" mice.

Male athymic "nude" mice (ANM) of the USC colony manifest spontaneous fasting hyperglycemia and reduced glucose tolerance; it has been proposed that they may represent a model of nonobese non-insulin-dependent diabetes. Following the recent demonstration that insulin secretion from the isolated, perfused pancreas of the male ANM appears to be hypersensitive to glucose, the function of individual pancreatic islet beta cells was investigated by measuring the membrane potential electrical activity. Initial studies demonstrated that the cyclic pattern of electrical activity in isolated female ANM islets is indistinguishable from that in control mouse islets. In contrast to control and female ANM beta cells, in which 11.1 mM glucose evoked approximately 50% maximal electrical activity, this concentration evoked almost 80% maximal activity in male ANM beta cells (p < 0.01). Investigating electrical responses at different glucose concentrations demonstrated that this increased sensitivity to glucose extends across the concentration range 2.8 to 22 mM. Assuming that in these islets, as in normal islets, electrical activity is associated with insulin release, these data indicate that the glucose-versus-insulin secretion dose-response is shifted to lower glucose concentrations at the level of the individual beta cell. Although this study demonstrates that altered beta-cell function occurs in the isolated islet of the male ANM, further investigation is under way to determine how the observed beta-cell glucose hypersensitivity is related to the hyperglycemia and impaired glucose tolerance that develop in these animals in vivo.

ATP-sensitive K channel modulation by products of PLA2 action in the insulin-secreting HIT cell line.

Insulin secretion from the islets of Langerhans may be initiated or potentiated by increased phospholipase A2 (PLA2) activity. This patch-clamp study of the insulin-secreting HIT tumor cell line assessed whether inhibition of the ATP-sensitive potassium (KATP) channel, which modulates the secretion-associated beta-cell electrical activity, contributes to the secretory response to PLA2. Exogenous PLA2 (100-1,000 mU/ml) reversibly suppressed KATP channel activity in excised inside-out patches. Similarly, mellitin (0.5-5 micrograms/ml), a bee venom component that increases phospholipid susceptibility to metabolism by PLA2, suppressed KATP channel activity, suggesting that PLA2 is present in excised patches. Adding low concentrations of particular lysophospholipids or arachidonic acid also reduced KATP channel activity in excised inside-out patches. In cell-attached patches, the lysophospholipids had a similar effect, whereas arachidonic acid caused channel stimulation; this latter effect was reversed by cyclooxygenase inhibitors. A recently identified ATP-stimulated PLA2 in beta-cells has been proposed as an important mediator of stimulus-secretion coupling in response to nutrients. The present data illustrating that initial products of PLA2 action on membrane phospholipids reduce KATP channel activity indicate a mechanism that may contribute an early stimulatory signal in this pathway. The observation that metabolism of arachidonate via the cyclooxygenase pathway causes KATP channel stimulation demonstrates a potential counterregulatory mechanism.

ATP mediates both activation and inhibition of K(ATP) channel activity via cAMP-dependent protein kinase in insulin-secreting cell lines.

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 microM range, but depressed it at higher concentrations. In inside-out patches, addition of the cAMP-dependent 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 microM in the presence of exogenous PKI progressively reduced the channel activity; it is proposed that this inactivation results from a reduction in kinase 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 microM), a nonhydrolyzable ATP analogue, AMP-PNP, enhanced the channel activity when present below 10 microM, 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.

Comparative study of K channel behavior in beta cell lines with different secretory responses to glucose.

The patch-clamp technique was used to identify and investigate two K channels in the cell membrane of the HIT cell, an insulin secreting cell line with glucose-sensitive secretion. Channel characteristics were compared with those of glucose-modulated K channels in the RINm5F cell, an insulin secreting cell line in which secretion is largely glucose insensitive. A 65.7 pS channel, identified with the ATP-sensitive K channel was seen in HIT cell-attached patches. Channel activity was dose-dependently inhibited by glucose, by 50 and 100% at 450 microM and 8 mM glucose, respectively, similar to the values previously reported for RIN cells. In inside-out patches channel activity was 50% inhibited by 56 microM ATP and completely blocked between 500 microM and 1 mM, again, similar to the values reported for RIN cells. As in RIN cells a second, considerably larger (184 pS), K channel was glucose sensitive; the glucose sensitivity was similar to that in RIN cells with 50 and 100% channel inhibition at 7.5 and 25 mM, respectively. After patch excision the mean channel conductance increased from 184 to 215 pS. Under these conditions activity was strongly calcium dependent in the range pCa 5-7, identifying this as a calcium- and voltage-dependent K (K(Ca, V] channel; the calcium sensitivity was similar to that of the adult rat beta cell K(Ca, V) channel. In inside-out RIN cell patches, the large K channel was less abundant but displayed a similar conductance (223 pS). However, its calcium sensitivity was more than 10 times lower than in HIT cells, similar to that of the K(Ca, V) channel in the neonatal rat beta cell, which also displays a reduced secretory response to glucose. Based on these observations, it is proposed that the low calcium sensitivity of the K(Ca, V) channel may be causally associated with secretory deficiency in RIN cells and the immature secretory response of the neonatal beta cell.

Metabolic regulation of the K(ATP) and a maxi-K(V) channel in the insulin-secreting RINm5F cell.

K channels in the cell membrane of the insulin-secreting RINm5F cell line were studied using the patch-clamp technique in cell-attached patch mode. With 140 mM K in the pipette, two channels displaying different conductive and kinetics properties were observed. A voltage-independent, inward-rectifying, 55-pS channel was active at rest (no glucose, -70 mV), but was almost completely inhibited by 5 mM glucose. A 140-pS channel was seen in the absence of glucose only after cell membrane depolarization with high (30 mM) K. This channel was voltage dependent, with a linear slope conductance between -60 and +60 mV, and was completely inhibited only by greater than 15 mM glucose. The former channel we identify as an ATP-sensitive channel previously described in excised patches and refer to it as the K(ATP) channel. The latter, because of its large conductance and voltage-dependent kinetics, will be referred to as the maxi-K(V) channel, adopting a nomenclature previously used to classify highly conductive K channels (Latorre, R., and C. Miller, 1983, Journal of Membrane Biology, 71:11-30). In addition to glucose, mannose and 2-ketoisocaproate, which also initiate insulin secretion and electrical activity in the islet beta cell, reduced the activity of both the K(ATP) and the maxi-K(V) channel. Lactate and arginine, which potentiate but do not initiate insulin secretion or beta cell electrical activity in normal islets, each caused a large reduction in maxi-K(V) channel activity, without consistently affecting the activity of K(ATP) channels. Another agonist that potentiates insulin secretion and electrical activity in normal cells, the tumor-promoting phorbol ester TPA, blocked maxi-K(V) channel activity while stimulating the activity of the K(ATP) channel, thereby implicating phosphorylation in the control of channel activity. These results indicate that metabolic substrates that initiate electrical activity and insulin secretion in normal beta cells reduce the activity of both the K(ATP) and the maxi-K(V) channel, while potentiating agents reduce only the maxi-K(V) channel. The possible role of these two channels in the processes of initiation and potentiation of the beta cell response is discussed.

Electrical activity, cAMP concentration, and insulin release in mouse islets of Langerhans.

The influence of forskolin and 3-iso-butyl-1-methylxanthine (IBMX) on mouse pancreatic beta-cell electrical activity, whole islet cAMP content, and insulin release were investigated. The two drugs potentiated to a similar extent both glucose-stimulated electrical activity and insulin release. In terms of the electrical response, both drugs potentiated the silent depolarization of the membrane in response to low (substimulatory) glucose concentrations, whereas at higher (stimulatory) glucose concentrations they caused an increase in the plateau fraction, with a response similar to the effect of increasing the glucose concentration. Both phases of insulin release were increased by each of the drugs. Ten micromolar forskolin and 100 microM IBMX caused an increase in intraislet adenosine 3',5'-cyclic monophosphate (cAMP) in the presence of 11.1 mM glucose, the former a 17-fold and the latter a 2-fold increase over the cAMP concentration in the presence of glucose alone. Because the two drugs lead to an increase in islet cAMP content, it is proposed that protein phosphorylation resulting from an activation of beta-cell cAMP-dependent protein kinases is responsible for the potentiation of the glucose-induced insulin release and beta-cell electrical activity. The observed effects on electrical activity are compatible with the hypothesis that cAMP-dependent phosphorylation induces alteration of the kinetics of the calcium-sensitive potassium permeability of the beta-cell plasma membrane. The increase in calcium entry into the beta-cell that would result from these alterations may be responsible for the cAMP-dependent potentiation of insulin release.

Electrical coupling between cells in islets of Langerhans from mouse.

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+.

Pancreatic beta-cell electrical activity: the role of anions and the control of pH.

The influence of chloride on the mouse pancreatic beta-cell membrane potential and the cell membrane mechanisms controlling intracellular pH (pHi) have been investigated using glass microelectrodes to monitor the membrane potential. It has been shown that chloride is distributed passively across the beta-cell membrane such that chloride potential is equal to the membrane potential. Withdrawal of perifusate chloride or bicarbonate and the application of the drugs 4-acetamido-4'-isethiocyanostilbene-2,2'-disulfonic acid (SITS) and probenecid, both blockers of transmembrane anion movement, have been used to establish that a chloride-bicarbonate exchange system is operative in the cell membrane and that it is one of the control mechanisms of pHi. Amiloride, a specific blocker of the transmembrane sodium proton exchange, has been used to demonstrate that this mechanism is also operative in the beta-cell membrane in the control of pHi. The hypothesis that the calcium-activated potassium permeability is proton sensitive at an intracellular site, a fall in pHi causing a fall in permeability and an increase in pHi causing an increase in permeability, has been used to explain many of the effects observed in this study.

Voltage noise measurements across the pancreatic beta-cell membrane: calcium channel characteristics.

1. Membrane potential fluctuations were measured in cells from mouse Islets of Langerhans identified as beta-cells by the characteristic pattern of electrical activity induced by 11 mM-D-glucose. 2. The membrane potential was controlled by adjusting the external potassium concentration, [K+]o, keeping the sum [Na+]o plus [K+]o constant. In the absence of glucose, when [K+]o is raised, the resulting depolarization is accompanied by a significant increase in voltage noise. 3 The amplitude and time course of the voltage noise were measured under various experimental conditions. The variance of the fluctuating voltage decreased monotonically along the depolarization induced by sudden increase in [K+]o, suggesting a monotonic reduction in the number of elementary events. 4. The frequency characteristics of the excess noise could be analysed as the sum of 1/f and 1/f2 components. While the 1/f component remained unaffected by the external application of 20mM-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 fc 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) omega (-1).