Article Figures & Data
Figures
- Fig. 1.
Action potential waveforms (APWs) recruit facilitation L-current in AC cells from calf but not adult cattle. To mimic Ca channel activation by depolarizing secretagogues such as ACh, we recorded APs in current clamp and played them back as voltage-clamp commands (APWs) in (A) calf and (B) adult bovine AC cells. Atleft, Ca currents elicited by single APWs (a1 ) before and after a train of 20 APWs delivered at 1 and 7 Hz in the same cell are shown; 4 min was allowed for recovery between trains. The superimposed current traces are (1) control Ca current and (2 and3) Ca current at the end of the 1 or 7 Hz train. Note that in A, APWs delivered at 1 Hz increased the Ca current by 48%, whereas at 7 Hz the Ca current was increased by 98%; in B no change was observed at either frequency, indicating the absence of facilitation. At right, Ca currents (b2 ) elicited by a single APW (b1 ) before and after three consecutive trains of 20 APWs at 7 Hz in the absence (1, 2) or presence of nisoldipine (1 μm), alone (3) or plus ω-CgTx (500 nm) + ω-AgaTx (10 nm) (4); a 4 min interval was allowed for recovery and application of antagonists. Nisoldipine selectively suppressed the increased Ca current brought about by the 7 Hz stimulation in calf (A) and decreased a component of Ca current (presumed “standard” L-type) in adult (B); the remaining current was almost completely suppressed by the addition of ω-CgTx + ω-AgaTx.
- Fig. 2.
Statistical analysis of Ca currents in AC cells from calf versus adult cattle: AP stimulation frequency and antagonist effects. A, Effect of AP stimulation frequency on the peak Ca current amplitude at the end of a train of 20 APWs at 1 or 7 Hz. Values are mean ± SEM; n is indicated inparentheses above each bar. *, Significantly different from the corresponding control at p < 0.0001.B, Pharmacological dissection of the Ca current components, evoked by APWs at 7 Hz, shows that facilitation L-type constitutes 42.32 ± 0.93% of the total Ca current in calf AC cells, whereas standard L-type contributes 34.1 ± 2.53% to the total current in adult AC cells.
- Fig. 3.
Distinct role of facilitation Ca channels in catecholamine secretion from calf AC cells. A, Calf AC cells were stimulated with trains of APs in current-clamp mode together with amperometric detection of catecholamine secretion. Amperometric spikes (Iamp) were generated in response to trains of single APs (Vm) evoked by short depolarizing current pulses at a frequency of 7 Hz (a1 ). Sequential trains of 490 APs at 7 Hz resulted in successive secretory episodes monitored as barrages of amperometric spikes; a 6 min rest period separated each train. As quantitated by the cumulative integral of the amperometric current (a3 ), the second round (Control 2) was always slightly increased relative to the first (Control 1), indicating that secretion shows no sign of exhaustion under these conditions (average increase in the second round was 12.8 ± 0.9%; n = 44). To determine whether facilitation Ca channels contribute to catecholamine secretion in calf AC cells, nisoldipine was added between trains. Secretion was substantially reduced under these conditions (a2, a3 , 44% inhibition compared with secretion in the first round). Inset(a4 ) shows expanded time base for the first 10 sec of AP-stimulated secretion. When facilitation Ca channels are recruited (Ctr1 and Ctr2), secretion starts within the first second of stimulation, whereas after nisoldipine (Nis) secretion only starts after a lag of ∼9 sec. B, Adult AC cells treated under conditions identical to those shown in A. Instead of enhancement, a slight depression in cumulative secretion was seen in the second round of stimulation (b3, Control 1, Control 2). When nisoldipine was applied before the second round of stimulation, the signal was further depressed (b2,b3, 37% inhibition) indicating that conventional L-type Ca channels contribute to secretion in these cells.Inset (b4 ) shows that all three types of Ca channels found in adult AC cells promote secretion, but with an average lag of 6 sec; after nisoldipine the lag is ∼9 sec.
- Fig. 4.
Strong stimulus–secretion coupling is predominant in calf AC cells but not in adult AC cells. To quantitate the delay between the onset of stimulation and detection of amperometric spikes from calf and adult AC cells, we analyzed latency histograms.A, In such histograms, “coupling” is defined as the degree of coincidence between APs and the subsequent amperometric spike(s). The time from the peak of the AP (vertical dashed line) to the beginning of a current spike (indicated here byarrows 1 and 2) is termed latency, and these are collected and displayed as latency histograms (Chow et al., 1992). B, Representative amperometric spikes (Iamp) generated from a calf AC cell in response to trains of single APs (Vm) evoked at 1 Hz illustrate the coupling between APs (bottom trace) and amperometric spikes (top trace). C, Latency histograms of multiple events collected from calf AC cells stimulated by single APs applied at 0.25 Hz (c1 ), 1 Hz (c2 ), or 7 Hz (c3 ), respectively. Filled barsrepresent “strongly coupled” signals with latency <25 msec, whereas open bars represent “weakly coupled” signals (latency 25–143 msec). At 0.25 Hz, nearly all amperometric spikes (97%) are strongly coupled; at 1 Hz, 83% are strongly coupled, whereas the remaining events form a dispersed tail. At 7 Hz, 40% of events are strongly coupled, whereas the other 60% are weakly coupled.D, Latency histograms from adult AC cells stimulated under conditions identical to those shown in C. No events were apparent at 0.25 Hz (d1 ), and only a plateau component of latencies from 0 to 143 msec was observed at 1 and 7 Hz (d2,d3). Only 4 and 14% of the total events had a latency <25 msec at 1 and 7 Hz, respectively. Latency histograms in C and D were determined from different cells stimulated by similar number of APs at each frequency, of which only 200 msec is shown 450 APs (c1, d1 , 0.25 Hz), 490 APs (c2, d2 , 1 Hz), and 490 APs (c3, d3 , 7 Hz). Only amperometric events having 50–90% rise time faster than 3 msec were selected for these histograms.
- Fig. 5.
Strong stimulus–secretion coupling is attributable to activation of facilitation L-type Ca channels. To determine the proximity of different types of Ca channels to release sites and their contribution to secretion at different frequencies, we analyzed latency histograms from amperometric experiments in the presence of antagonists that selectively block L-type (nisoldipine) or N-type [ω-Conotoxin-GVIA (ω-CgTx)] and P-type [ω-Agatoxin-IVA (ω-AgaTx)] Ca channels. A, Nisoldipine eliminates the strongly coupled peak of secretion at all frequencies in calf AC cells. Latency histograms were derived from the amperometric spikes that resulted from two successive stimulation periods of 1 Hz (a1, a2 , 490 APs each) or 7 Hz (a3, a4 , 490 APs each) in the absence (Control, a1, a3 ) or presence of nisoldipine (Nisoldipine, a2, a4 ). At 7 Hz, nisoldipine suppressed 85% of events with latencies <25 msec and 30% of those with latencies >25 msec after the AP (49% of total events). In parallel experiments (a5 , >490 APs), the combination of ω-CgTx and ω-AgaTx had little effect on the short-latency responses (reduced 14% of events <25 msec) but eliminated 77% of events from the weakly coupled plateau (53% of total events). B, Latency histograms derived from adult AC cells using an experimental protocol similar to that shown in A: 1 Hz (b1, b2 , 490 APs), 7 Hz (b3, b4 , 490 APs) stimulation, each set from the same cell, in the absence (Control, a1 anda3 ) or presence of nisoldipine (Nisoldipine, b2, b4 ), or 7 Hz (b5 ) in the presence of ω-CgTx + ω-AgaTx. At 7 Hz, in comparison with the control, 30 and 69% of total events were suppressed by nisoldipine or the combination of toxins, respectively.
- Fig. 6.
Estimated distance between facilitation Ca channels and the adjacent release site. To estimate δ from the decay time constant (τ) of an amperometric latency histogram (ALH), an empirical function was obtained by a standard optimum fit procedure of data describing the relationship between Ca channels and secretory sites in adult AC cells (Klingauf and Neher, 1997). In that model, two points (τ = 8.7 msec, δ = 30 nm; τ = 25 msec, δ = 300 nm) were calculated from a specific set of conditions prevalent in those cells (■). To derive the empirical function, we added a third point (δ = 10 nm, τ = 1 msec; ▪) derived from work on synaptic terminals (Adler et al., 1991; Lando and Zucker, 1994; Roberts, 1994; Sabatini and Regehr, 1996). An optimum fit with the three points (τ = 1, δ = 10; τ = 8.7 msec, δ = 30 nm; τ = 25 msec, δ = 300 nm) results in an empirical formula log δ = 0.94 + 0.063 τ. The resultant equation was plotted, and the known τ value of 3.1 msec derived from the latency histogram at 0.25 Hz (Table1) was used to determine the corresponding δ value of 13 nm by interpolation (•). The triangles show modified data points of Klingauf and Neher (1997), corrected for 1000 pA Ca current (▴) (see Results for details).
Tables
- Table 1.
Kinetic properties of catecholamine release in AC cells from calf versus adult cattle: pharmacological dissection
Condition τ (msec) % Events ≤25 msec % Events ≥25 msec IntegralQamp (pC) Rate (pC/sec) Calf, 0.25 Hz, control (n = 19) 3.18 ± 0.08 93.3 ± 1.05a 6.7 ± 1.05 Calf, 1 Hz, control (n = 144) 2.92 ± 0.08 78.72 ± 0.68a 21.28 ± 0.68 Calf, 1 Hz, nisoldipine (n = 13) 15.13 ± 2.23 84.83 ± 2.21 Calf, 7 Hz, control (n = 109) 3.16 ± 0.08 40 ± 0.1a 60 ± 0.1 374.7 ± 9.97b,c,d 5.39 ± 0.16b,c,d Calf, 7 Hz, nisoldipine (n = 20) 14.75 ± 1.13 85.25 ± 1.13 216 ± 13b 3.35 ± 0.25b Calf, 7 Hz, ω-CgTx + ω-AgaTx (n = 12) 3.38 ± 0.27 70 ± 0.51a 30 ± 0.51 174 ± 3.4d 2.53 ± 0.1d Adult, 1 Hz, control (n= 20) 10.2 ± 1.4 89.8 ± 1.4 Adult, 1 Hz, nisoldipine (n = 9) 13.56 ± 0.83 86.44 ± 0.83 Adult, 7 Hz, control (n = 22) 14.1 ± 0.75 85.9 ± 0.75 203.4 ± 12.3c 2.99 ± 0.19c Adult, 7 Hz, nisoldipine (n = 13) 12.2 ± 0.9 87.8 ± 0.9 131.3 ± 8.3b,c,d 1.91 ± 0.12b,c,d Adult, 7 Hz, ω-CgTx + ω-AgaTx (n = 12) 14 ± 0.4 86 ± 0.4 τ, % of events <25 msec, and % of events >25 msec are calculated from the individual amperometric delay histograms (ALHs) in each condition. τ is obtained from a monoexponential fit of the “strongly coupled” events in each histogram. IntegralQamp = Iampdt is calculated from the amperometric traces (70 sec) under the different conditions described in the table. The integral rate corresponds to the slope of a linear fit of the amperometric integral.
a,b,c,dThese values are significantly different from each other at p < 0.0001. Measurements are given as means ± SEM.
