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PubMed1992

Synergistic action of cyclic GMP on catecholamine-induced chloride current in guinea-pig ventricular cells.

1. Effects of cyclic GMP on the catecholamine-induced chloride current (ICl) were studied using the whole-cell patch-clamp technique combined with internal perfusion in single ventricular myocytes dispersed from guinea-pig heart. 2. When ICl was activated by submaximal doses of isoprenaline (0.01-0.1 microM), adrenaline (0.5-1 microM) and histamine (0.2-0.5 microM), intracellular dialysis with cyclic GMP (10-100 microM) induced an extra increase of ICl. No further increase of ICl was induced by cyclic GMP when ICl was maximally activated. In the absence of agonists, cyclic GMP failed to induce ICl. 3. The enhancement by cyclic GMP was also observed when ICl was activated by external application of 0.2-1.0 microM-forskolin or by internal dialysis with a pipette solution containing 50-200 microM-cyclic AMP. 4. In contrast to cyclic GMP, 10-1000 microM-dibutyryl cyclic GMP and 8-bromo-cyclic GMP were ineffective in modifying ICl. 5. Milrinone (1-10 microM), a specific inhibitor of a kind of phosphodiesterase which is inhibited by cyclic GMP, also enhanced ICl activated by submaximal doses of isoprenaline. Milrinone itself did not activate ICl. 6. When ICl was enhanced by 5 microM-milrinone, an additional application of cyclic GMP failed to increase ICl. In the presence of cyclic GMP, milrinone failed to enhance ICl. 7. The above findings on ICl are analogous to the enhancement by cyclic GMP of the beta-adrenergic stimulation of the Ca2+ current reported in the same preparation, and support the hypothesis that in mammalian cardiac cells cyclic GMP potentiates elevation of cyclic AMP induced by beta-adrenergic agents, and thereby increases the amplitudes of ionic currents.

Animals↗
PubMed1992

Inward rectification of acetylcholine-elicited currents in rat phaeochromocytoma cells.

1. Currents elicited by acetylcholine (ACh) were studied in the rat phaeochromocytoma cell line, PC12, using patch-clamp techniques. 2. Whole-cell ACh-elicited currents are inwardly rectifying and intracellular Mg2+ can play a role in determining the extent of whole-cell current rectification. Increasing the intracellular Mg2+ concentration, [Mg2+]i, augmented the rectification. The effects of increased [Mg2+]i on the whole-cell current can be explained by the block of receptor channels by Mg2+. 3. In the nominal absence of internal divalent cations, however, a substantial degree of rectification remains. This rectification is probably not due to divalent cations, as buffering the external Mg2+ concentration to 50 microM and the internal concentration to nominally 0 Mg2+ did not reduce the rectification. The remanent rectification was not due to block by the main permeant cation, Na+. Using K+ or Cs+ as the main monovalent cation inside the cell did not diminish the rectification. Neither replacing the pH buffer, HEPES, with phosphate buffer nor increasing the intracellular pH removed the rectification. 4. For ACh receptor channels in excised patches, the voltage dependence of the probability of being open (Popen) stemmed mainly from the voltage dependence of the channel burst duration. The channel opening rate was relatively voltage independent. The weak voltage dependence displayed by the channel burst duration was insufficient to account for the reduced whole-cell outward current at positive potentials. The mean burst duration of the channel did not have a simple logarithmic relationship with voltage. 5. In the absence of intracellular Mg2+, the instantaneous current-voltage relationship for whole-cell currents was linear suggesting that the I-V relationship of single channels in perfused cells is linear and does not contribute to the rectification of the whole-cell current. 6. In perfused cells, receptor channels had a low steady-state probability of being open at positive potentials compared to channels in excised patches. Voltage jumps to positive potentials revealed a process in perfused cells which could account for the low Popen. Relaxations of agonist-induced current at +40 mV had a large, exponentially decaying component that quickly closed channels (rate constant, tau, approximately 400 microseconds). The mechanism responsible for this decay could explain the rectification that remains in the absence of intracellular divalent cations.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholine↗
PubMed1992

The inward rectifier K+ current underlies oscillatory membrane potential behaviour in bovine pigmented ciliary epithelial cells.

1. Fresh bovine, pigmented ciliary epithelial cells possess an inward rectifier current activated by hyperpolarization. This current was investigated using whole-cell patch-clamp techniques. At the holding potential of -70 mV, and with EK (potassium equilibrium potential) set at -84 mV, a small outward current flowed through the inward rectifier that was sensitive to external K+, becoming more outward in 0.5 mM K+ and progressively more inward in 20 and 50 mM K+. 2. The inward rectifier showed V-EK dependence; increasing [K+]o increased the inward conductance from 1.28 nS in 5 mM K+ to 7.42 nS in 50 mM K+. The conductance at a given V-EK was proportional to the square root of [K+]o. 3. It was blocked by external Cs+ but replacing K+ in the pipette with Cs+ blocked only outward ion movement through the inward rectifier. Inward rectification was also blocked by Ba2+ (85% with Ki (concentration giving half-maximal inhibition) = 3.1 x 10(-5) M) and TEA+ (74% with Ki = 2.9 x 10(-4) M). 4. The activation time constant was voltage dependent, decreasing from 5 ms to 0.7 ms over the voltage range -90 to -170 mV. With increasing hyperpolarization the current exhibited time-dependent decay. The time constant for this process was voltage sensitive but the steady-state inactivation was independent of external [K+]. 5. The current disappeared in culture within 8 days. 6. Solution flow over the cell inactivated the inward rectifier, a property that may be related to [K+]o. 7. In current clamp the cells exhibited an unstable region at a potential of around -70 mV. Once in this region oscillations and regenerative hyperpolarizing potentials were observed. This behaviour was eliminated by treatments that blocked (Cs+, Ba2+) or removed (0.5 mM K+) active inward rectification. 8. It is suggested that these oscillations may represent a process of cation loading, the first step in the secretion of aqueous humour.

Animals↗
PubMed1992

Whole-cell chloride conductances in cultured brushed human nasal epithelial cells.

Human airway epithelial cells were obtained by nasal brushing, thus avoiding the use of proteolytic enzymes for cell isolation. Whole-cell Cl- conductances were studied in these cells by means of the patch-clamp technique. During whole-cell recordings, cell swelling activated a Cl- conductance that was blocked by indanyloxyacetic acid (48 +/- 10% inhibition at 50 microM). The swelling-induced current outwardly rectified and showed inactivation at depolarizing voltages (> or = +60 mV) and activation at hyperpolarizing voltages (< or = -30 mV). The voltage sensitivity of current activation was approximately twice that of inactivation. Another Cl- current with different kinetics was observed when nonswollen airway cells were stimulated with ionomycin (2 microM) in the presence of 1 mM Ca2+. The Ca(2+)-induced current exhibited activation during depolarizing voltage steps (> or = +40 mV) and inactivation during hyperpolarizing voltage steps (< or = -40 mV). In contrast to the swelling-induced current, the activation of Ca(2+)-induced current was less sensitive to voltage compared with its inactivation. Tail current analysis suggested that Cl- channels having a linear current-voltage relation mediate the response to Ca2+. This study indicates that brushed human nasal epithelial cells possess Cl- conductances that are regulated by cell swelling and Ca2+ and that they represent a useful in vitro model for studying ion transport in epithelia.

Calcium↗
PubMed1992

Anion channels for amino acids in MDCK cells.

Large losses of amino acids by diffusion were previously observed in Madin-Darby canine kidney (MDCK) cells during volume regulation. Also, an outward rectifying anion channel was activated. Because this channel was not selective among anions, it was suggested that it could be permeable to amino acids. Its permeability to aspartate, glutamate, and taurine was studied using the patch-clamp technique in the inside-out configuration. Solutions containing 500 mM aspartate or glutamate were used on the cytoplasmic side of excised patches to detect single-channel currents carried by these anions. Permeability ratios were estimated in two different ways: 1) from the shift in reversal potential of current-voltage curves after anion replacement in the bath solution and 2) from comparisons of amplitudes of single-channel currents carried by tested anions and chloride, respectively. The values of aspartate-to-chloride and glutamate-to-chloride permeability ratios obtained with both methods were quite consistent and were of the order of 0.2 for both amino acids. Taurine in solutions at physiological pH 7.3 is a zwitterionic molecule and bears no net charge. To detect single-channel currents carried by taurine, solutions containing 500 mM taurine at pH 8.2 were used in inside-out experiments. Under these conditions 120 mM of negatively charged taurine was present in the solutions bathing the cytoplasmic side of excised patches. The permeability ratio estimated from the shift in reversal potential was 0.75. These results showed that some of the organic compounds released by cells during regulatory volume decrease could diffuse through this outwardly rectifying anionic channel.

Amino Acids↗
PubMed1992

Beta-adrenergic regulation of H+ secretion by cultured outer medullary collecting duct cells.

The effects of the beta-adrenergic agonist isoproterenol (Iso) on cells of the inner stripe portion of the rabbit outer medullary collecting duct (OMCDi) grown in primary culture were examined using whole cell patch-clamp techniques and measurements of intracellular pH (pHi) and Ca2+. Iso (10(-6) M) increased the cellular Cl- conductance, and this effect was mimicked by treatment of the cells with dibutyryladenosine 3',5'-cyclic monophosphate (cAMP, 10(-5) M) or protein kinase A (PKA, 0.4 U/ml). Iso did not alter the baseline pHi, but it did increase the activity of both the Cl-/HCO3- antiporter and the H(+)-adenosinetriphosphatase (H(+)-ATPase). The increase in Cl-/HCO3- antiporter rate was mimicked by dibutyryl-cAMP plus 3-isobutyl-1-methylxanthine (cAMP + IBMX, 10(-4) M + 10(-5) M). However, the Iso-induced stimulation of the H(+)-ATPase activity was not mimicked by cAMP + IBMX. Measurements of intracellular Ca2+ showed that Iso also increased intracellular Ca2+ levels. This response was not dependent on extracellular Ca2+, nor did cAMP + IBMX appreciably alter intracellular Ca2+. Consequently, we postulate that beta-adrenergic agonists are potential stimulators of OMCDi H+ secretion. These agonists stimulate cellular HCO3- efflux through a signal transduction pathway involving cAMP and PKA. However, a different signal transduction pathway appears to mediate the stimulation of cellular H+ efflux. This second pathway may involve an elevation of intracellular Ca2+.

1-Methyl-3-isobutylxanthine↗
PubMed1992

Calcium-activated chloride channels in cultured embryonic Xenopus spinal neurons.

1. Single-channel currents were recorded from Xenopus spinal neurons developing in vitro using the patch-clamp technique, to identify the channels underlying the large and small macroscopic Ca(2+)-activated Cl- currents (ICl(Ca)) present in these cells. 2. Channels of large (maxi-channels; 310 pS) and smaller conductance (mini-channels; 50-60 pS) are activated by elevation of cytoplasmic Ca2+ concentration. Channel activity is not altered by subsequent removal of Ca2+ from the bath, arguing against a direct ligand-type Ca2+ dependence. The much higher incidence of channel activation in cell-attached patches from cells permeabilized with the Ca2+ ionophore A23187 than in excised patches also suggests the involvement of some unidentified intracellular factor. 3. The reversal potential of maxi-Cl- channels is not altered by changes in Na+ concentration, but is shifted in the negative direction by the substitution of Cl- by methanesulfonate on the intracellular side of the patch, indicating their anionic selectivity. 4. Maxi-Cl- channels exhibited the presence of multiple probable subconductance states and showed marked voltage-dependent inactivation above and below +/- 20 mV. 5. Examination of maxi-Cl- channels at early times in culture (6-9 h) and 24 h later did not reveal any developmental change in the characteristics described above. However, the mean open duration of the channel was found to increase twofold during this period of time. 6. The simultaneous presence of maxi- and mini-Cl- channels prevented detailed characterization of the latter. The anionic selectivity of mini-Cl- channels is suggested by their reversal potential that lies close to the Cl- equilibrium potential.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗
PubMed1992

Calcium currents in solitary hair cells isolated from frog crista ampullaris.

Some properties of Ca2+ currents in hair cells isolated from frog semicircular canals by enzymatic or mechanical treatment were studied by using the whole-cell configuration of the patch-clamp technique. After blocking the large outward K+ currents by substituting Cs+ for K+ and adding tetraethylammonium to the pipette filling solution, voltage- and time-dependent inward currents were clearly detectable in the presence of 4 mM Ca2+ in the extracellular solution. Ca2+ current was recruited at test potentials more positive than -60 mV, showed a rapid activation, and exhibited no inactivation during 150-ms depolarizing pulses. The maximal amplitude was attained at about -20 mV, with an average value of about 80 pA. When Ca2+ in the extracellular solution was replaced with Ba2+, the magnitude of inward currents increased about twofold. Ba2+ currents were blocked more effectively by Cd2+ than by Ni2+, were suppressed by 0.5 microM omega-conotoxin, and were virtually unaffected by amiloride. The dihydropyridine Bay K 8644 caused a marked voltage-dependent increase in inward currents. The present data suggest that hair cells from frog crista ampullaris are endowed with a homogeneous population of Ca2+ channels having several properties similar to those described for neuronal L channels. Since these channels are recruited in a range of potentials close to the resting level, it is suggested that they subserve the control of both resting and evoked transmitter release from the basal pole of the hair cells.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1992

The effect of ATP on Ca(2+)-dependent K+ channels of human red cells.

The action of ATP on Ca(2+)-dependent K+ channels was studied in fresh human erythrocytes using patch-clamp techniques. Single-channel current was recorded at pH 6.5 from inside-out patches in the presence of symmetrical K+ gluconate solutions, containing both 1 microM free Ca2+ in the bath and 0.5 mM LaCl3 on the pipette side. With no ATP, the electrical activity revealed low-conductance K+ channels (25 pS), which showed inward rectification and an opening kinetics dependent on membrane potential. When ATP (1 mM) and Mg2+ (2 mM) were added together and a depolarizing potential was simultaneously applied, only a high-conductance channel (about 75 pS) was observed. This channel showed no rectifying properties and it was not found if ATP was added in the absence of Mg2+. Channel activity was enhanced by adding fluoride (10 mM) or trifluoperazine (50 microM) whilst it was reduced after incubating with dibutyryl cAMP (50 microM) or alkaline phosphatase (250 U/ml). On the other hand, when fragmented membranes from inside-out vesicles were incubated with gamma-32 P-ATP and 1 microM free Ca2+ under above conditions, only two high-molecular weight polypeptides (235 and 320 kDa) were labelled with 32P. The results suggest that ATP-mediated phosphorylation of Ca(2+)-dependent K+ channels leads to a high-conductance state.

Adenosine Triphosphate↗
PubMed2026

Biophysical mechanisms underlying the generation and maintenance of rule-learning engram.

Training rodents in a particularly difficult olfactory-discrimination task results with acquisition of high-skill to perform the task superbly, termed 'rule-learning'. We show that rule-learning occurs abruptly, in a "light-bulb moment". Using whole-cell patch-clamp recordings from the piriform cortex (PC) of Fos2A-iCreER/TRAP2 mice, we target activated-neurons, expressing immediate early genes (IEG). We notice, from the onset of training, IEG-positive neurons from trained animals display enhanced intrinsic excitability. Subsequently, synaptic excitation and inhibition are enhanced in these neurons, in a coordinated, cell-wide process. Additionally,&#xa0;in parallel, we detect&#xa0;the density of IEG-expressing neurons sharply declines. Double labeling with TRAP and c-Fos reveal that nearly two-thirds of the rule-memory cell ensemble neurons are activated from the beginning of training. Silencing TRAP-expressing neurons using inhibitory DREADD leads to a complete loss of rule memory. Hence, we propose that rule learning occurs at a discrete moment and is developed through a gradual process that stabilizes the memory of the rule.

Animals↗
PubMed2026

Automated patch clamp data improve variant classification and penetrance stratification for SCN5A-Brugada syndrome.

BACKGROUND AND AIMS: Brugada Syndrome (BrS) is an inherited arrhythmia disorder that causes an elevated risk of sudden cardiac death. Approximately 20% of patients with BrS have rare variants in SCN5A, which encodes the cardiac sodium channel NaV1.5. Genetic workup of BrS is often complicated by SCN5A variants of uncertain significance (VUS) and/or incomplete penetrance. This study deployed an SCN5A-BrS functional assay at cohort scale to facilitate the implementation of genetic and precision medicine. METHODS: All 252 missense and in-frame insertion/deletion SCN5A variants from a previously published large cohort of BrS cases (n = 3335 patients) were analysed using a calibrated high-throughput automated patch-clamp (APC) assay. Variant functional Z-scores were assigned evidence levels ranging from BS3_moderate (normal function) to PS3_strong (loss-of-function), as defined by American College of Medical Genetics and Genomics criteria. Functional evidence was combined with population frequency, hotspot, case counts, protein-length changes, and in silico predictions. Odds ratios of BrS case-control enrichment and penetrance for BrS were calculated from variant frequencies in the BrS cohort and in gnomAD. RESULTS: Most variants (146/252) were functionally abnormal (Z &#x2264; -2), with 100 having severe loss-of-function (Z &#x2264; -4). Functional evidence enabled the reclassification of 110 of 225 VUS; 104 to likely pathogenic and 6 to likely benign. SCN5A variants with loss-of-function were mainly localized to the transmembrane domains, especially the regions comprising the central pore. SCN5A variant penetrance was proportional to the severity of loss-of-function; variants with Z &#x2264; -6 had penetrance of 24.5% (15.9%-37.7% CI) and an odds ratio of 501 for BrS. CONCLUSIONS: This cohort-scale APC dataset stratifies SCN5A variants found in BrS patients into normal function 'bystander' variants that have a low risk of BrS and loss-of-function variants that have a high risk for BrS. Functional data can be integrated with other criteria to reclassify a substantial fraction of VUS. The dataset helps clarify the SCN5A-BrS relationship and will improve the diagnosis and clinical management of BrS probands and their families.

Humans↗
PubMed1992

Stretch-activated non-selective cation channels in the antiluminal membrane of porcine cerebral capillaries.

1. Single stretch-activated (SA) channels have been studied in isolated brain capillary endothelial cells as well as in the antiluminal membrane of intact porcine cerebral capillaries using the patch-clamp recording technique. 2. The SA channels were found to be cation selective and permeable to Na+, K+, Ba2+ and Ca2+. 3. With monovalent cations in the patch pipette, the channels showed inward rectification in cell-attached patches with a single-channel conductance of 37 pS at negative and 24 pS at positive clamp potentials. 4. With either 70 mM-Ca2+ or Ba2+ in the patch pipette, the current-voltage relation was linear with slope conductances of 16 and 19 pS, respectively. 5. Mean channel open probability increased with increasing pressure and with depolarizing clamp potentials. 6. Cell swelling induced by hypotonic shock activated the SA channels in cell-attached experiments. 7. The SA channel may be involved in cell volume or blood flow regulation. The contribution of these channels to the regulation of cerebrospinal salt and water content, especially in brain oedema, is discussed.

Animals↗
PubMed1992

Structural and functional crosstalk between acetylcholine receptor and its membrane environment.

Nicotinic acetylcholine receptor (AChR) is a transmembrane protein belonging to the superfamily of rapid, ligand-operated channels. Theoretical models based on thermodynamic criteria assign portions of the polypeptide chains to the lipid bilayer region. From an experimental point of view, however, the relationship between the two moieties remains largely unexplored. Current studies from our laboratory are aimed at defining the structural, dynamic, and functional relationship between membrane lipids and AChR. We are particularly interested in establishing the characteristics of and differences between the lipids in each leaflet of the bilayer and the belt or "annular" lipids immediately surrounding AChR and the bulk bilayer lipids. We are also interested in determining the possible implications of lipid modifications on AChR channel properties. Toward these ends, fluorescence and other spectroscopic techniques, together with biochemical analyses and patch-clamp studies, are currently being undertaken. Correlations can be established between structural aspects of phospholipid packing in the immediate perimeter of AChR and other properties of these annular lipids revealed by dynamic spectroscopic and molecular modeling techniques. Lipid compositional analyses of the clonal muscle cell line BC3H-1 and chemical modification studies have been carried out by incubation of intact cells in culture and of membrane patches excised therefrom with liposomes of different lipid composition. These studies have been combined with electrophysiological measurements using the patch-clamp technique, with the aim of determining the possible effects of lipids on the channel properties of muscle-type AChR. A variety of experimental conditions, involving polar head and fatty acyl chain substitution of phospholipids and cholesterol incorporation, are being assayed in the BC3H-1 cells.

Acetylcholine↗
PubMed1992

Functional and immunological responses of Jurkat lymphocytes transfected with the substance P receptor.

1. We have transfected the rat substance P receptor (SPR) cDNA into the leukemic T-lymphocyte cell line Jurkat (J-wt) in order to study the effects of substance P (SP) on lymphocyte signaling mechanisms and the resultant neuropeptide-induced immunological changes. 2. The SPR cDNA was transfected into J-wt by the method of electroporation. Clones expressing SPRs were selected using a functional assay that measured SP-induced mobilization of intracellular Ca2+ ([Ca2+]i) in a fluorescence activated cell sorter (FACS) and by their expression of specific 125I-SP binding. 3. One clone, J-SPR, was identified and shown by Northern blot and 125I-SP saturation binding techniques to express the 2.2-kb SPR message and approximately 50,000 SPRs/cell with a Kd of 0.3 nM, respectively. Stimulation of J-SPR by SP resulted in the rapid mobilization of [Ca2+]i. This response was dose dependent in the range 10(-11)-10(-6) M SP and was maximal at 10(-7) M SP, with an EC50 of 0.3-0.5 nM SP. We further demonstrated that the SPR is rapidly desensitized following SP stimulation and by activation of the cell's T-cell receptor (TCR). Whole-cell patch-clamp experiments on J-SPR show that SP stimulation induces a Cl- current by a Ca2+ mediated process dependent on Ca2+/calmodulin-dependent protein kinase (CaMK). 4. Stimulation of J-SPR by SP results in changes in the cell surface expression of a number of molecules that play important roles in cell adhesion and activation: the expression of LFA-1 is decreased, and CD2 and IL-2 receptors are increased by 30 min, 6 hr, and 24 hr, respectively, following stimulation, as assessed by antibody staining in a FACS. 5. The expression of functional SPRs in Jurkat lymphocytes will not permit a detailed examination of how the activation of SPRs result in altered immune responses and further elucidate the role this neuropeptide receptor plays in inflammation.

Calcimycin↗
PubMed1992

Characterization of the effects of a new Ca2+ channel activator, FPL 64176, in GH3 cells.

We examined the effects of the benzolpyrrole-type Ca2+ channel activator FPL 64176 on voltage-dependent L-type Ca2+ channels in rat anterior pituitary (GH3) cells. FPL 64176 increased K(+)-dependent Ca2+ influx into GH3 cells with an EC50 value of 1.2 x 10(-7) M but had no effect on the binding of [3H]PN200-110 to GH3 cell membranes at concentrations up to 10(-6) M. Whole-cell patch-clamp electrophysiology revealed that FPL 64176 (1 microM) increased L-type Ca2+ channel current amplitude and shifted the current-voltage relationship in the hyperpolarizing direction. Furthermore, Ca2+ channel current activation and deactivation were prolonged. Single-channel analysis showed that FPL 64176 increased both the probability of channel opening and the mean channel open time. Interestingly, the effect of FPL 64176 on channel open time was highly voltage dependent, with much longer openings being observed at more hyperpolarized potentials. We conclude that FPL 64176 represents a new class of L-type Ca2+ channel activator with a novel site and mechanism of action.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1992

Developmental changes in the beta-adrenergic modulation of calcium currents in rabbit ventricular cells.

We studied the developmental changes in the beta-adrenergic modulation of L-type calcium current (ICa) in enzymatically isolated adult (AD) and newborn (NB, 1-4-day-old) rabbit ventricular cells using the whole-cell patch-clamp method. ICa was measured as the peak inward current at a test potential of +15 mV by applying a 180-450-msec pulse from a holding potential of -40 mV with Cs(+)-rich pipettes and a K(+)-free bath solution at room temperature. In control, ICa density (obtained by normalizing ICa to the cell capacitance) was significantly higher in AD cells (5.5 +/- 0.2 [mean +/- SEM] pA/pF, n = 65) than in NB cells (2.6 +/- 0.1 pA/pF, n = 60). Isoproterenol (ISO, 1 nM-30 microM) increased ICa in a dose-dependent manner for both groups. The maximal effect (Emax) of ISO, expressed as percent increase in ICa over control levels, and the concentration for one half of the maximal effect (EC50) were 203% and 51 nM, respectively, for AD cells and 111% and 81 nM, respectively, for NB cells. The effect of ISO (1 microM) on ICa was decreased as the test potential was increased from -10 to +40 mV. However, the ratio of the percent increase in ICa for AD versus NB cells was almost constant (2.09-2.45) at each test potential. Dose-response curves of forskolin (FOR, 0.3-50 microM) gave Emax and EC50 of 268% and 0.74 microM, respectively, for AD cells and 380% and 1.15 microM, respectively, for NB cells. After stimulating ICa by 10 microM ISO, the addition of 10 microM FOR produced a further increase in ICa of only 12 +/- 2% in AD cells (n = 4) but a further increase of 140 +/- 41% in NB cells (n = 6). FOR (10 microM) did not produce any increase in ICa for AD and NB cells after stimulating ICa by intracellular application of 200 microM cAMP. ICa density stimulated by 10 microM ISO (17.8 +/- 1.1 pA/pF, n = 7), 10 microM FOR (21.0 +/- 1.3 pA/pF, n = 8), or 200 microM cAMP (18.0 +/- 1.3 pA/pF, n = 5) was equivalent in AD cells, whereas ICa density stimulated by 10 microM ISO (5.8 +/- 0.6 pA/pF, n = 9) was significantly lower than that stimulated by either 10 microM FOR (13.8 +/- 1.5 pA/pF, n = 7) or 200 microM cAMP (13.4 +/- 0.7 pA/pF, n = 7) in NB cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗