Kappa-opioid receptor activation modulates Ca2+ currents and secretion in isolated neuroendocrine nerve terminals

Abstract

Whole-cell patch-clamp recordings were performed together with time-resolved measurements of membrane capacitance (Cm) in nerve terminals acutely dissociated from neurohypophysis of adult rats to investigate modulation of Ca2+ currents and secretion by activation of opioid receptors. Bath superfusion of the kappa-opioid agonists U69,593 (0.3-1 microM), dynorphin A (1 microM), or U50,488H (1-3 microM) reversibly suppressed the peak amplitude of Ca2+ currents 32. 7 +/- 2.7% (in 41 of 56 terminals), 37.4 +/- 5.3% (in 5 of 8 terminals), and 33.5 +/- 8.1% (in 5 of 10 terminals), respectively. In contrast, tests in 11 terminals revealed no effect of the mu-opioid agonist [D-Pen2,5]-enkephalin (1-3 microM; n = 7) or of the delta-agonist Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol (1 microM; n = 4) on Ca2+ currents. Three components of high-threshold current were distinguished on the basis of their sensitivity to blockade by omega-conotoxin GVIA, nicardipine, and omega-conotoxin MVIIC: N-, L-, and P/Q-type current, respectively. Administration of U69,593 inhibited N-type current in these nerve terminals on average 32%, whereas L-type current was reduced 64%, and P/Q-type current was inhibited 28%. Monitoring of changes in Cm in response to brief depolarizing steps revealed that the kappa-opioid-induced reductions in N-, L-, or P/Q-type currents were accompanied by attenuations in two kinetically distinct components of Ca2+-dependent exocytotic release. These data provide strong evidence of a functional linkage between blockade of Ca2+ influx through voltage-dependent Ca2+ channels and inhibitory modulation of release by presynaptic opioid receptors in mammalian central nerve endings.

Fig. 1.

Opioid-induced inhibition of Ca²⁺ currents mediated via κ-opioid receptors.A–C, Recordings from three different terminals of whole-terminal Ca²⁺ currents evoked by a 100 msec (A, C) or 50 msec (B) step to +10 mV from −90 mV before the application and in the presence of selective κ-opioid (dynorphin A, 1 μm; U69,593, 1 μm), δ-opioid (DPDPE, 1 μm), or μ-opioid (DAMGO, 3 μm) agonists. Only the κ-opioid agonists reversibly inhibited Ca²⁺ currents. D₁,D₂, Recordings from another terminal showing that this effect of U69,593 was blocked by the κ-selective antagonist nor-BNI (1 μm). E, Comparison of effects of κ-, μ-, and δ-opioid-selective drugs on Ca²⁺ current. Open bars represent the mean ± SEM of the values obtained in the presence of a particular agonist (number of terminals shown in denominator); values are expressed as a percentage of the predrug control current. Current amplitudes were reduced significantly (p < 0.05) by U69,593, dynorphin A, and U50,488H but not by DAMGO and DPDPE. Filled barsrepresent only the data obtained from opioid-responsive terminals (number shown in numerator) and demonstrate equivalent inhibitory efficacy of the three κ-opioid agonists examined.

Fig. 2.

κ-Opioids inhibited GVIA-sensitive, nicardipine-sensitive, and MVIIC-sensitive Ca²⁺current components. A, upper, Recordings from a single nerve terminal show the effects of U69,593 (traces marked with asterisks) on Ca²⁺ currents evoked by 50 msec steps to +10 mV from a holding potential of −90 mV before the application and in the presence of Ca²⁺ channel-type selective blockers. Sequential administration of GVIA (0.5 μm), nicardipine (10 μm), and MVIIC (1 μm) completely abolished whole-terminal current. The ability of U69,593 to suppress Ca²⁺ current was greatly reduced after application of each of these blockers. A, lower, Subtraction of the corresponding currents evoked before and after application of antagonists yielded pharmacologically distinguished N-, L-, and P/Q-type current components in control conditions and in the presence of U69,593. B, Relative contribution of pharmacologically distinguished N-, L-, and P/Q-type current components to the total whole-terminal current and their sensitivity to the inhibitory effect of U69,593. Bars in the graph represent the mean ± SEM of the normalized current amplitudes obtained from the number of terminals shown, examined using the experimental protocol depicted in A.

Fig. 3.

Comparison of current–voltage and ΔC_m–voltage relationships determined for single nerve endings. C_m responses (A) and corresponding Ca²⁺currents (B) evoked in a single terminal by 200 msec step depolarizations to different command potentials (shown near corresponding traces) from a holding potential of −90 mV. The depolarizations to −10 and +50 mV produced small currents that were accompanied by an immediate, step-likeC_m increase, whereas steps to +10 mV evoked a larger current and prolonged C_m increase lasting for several seconds. Ca²⁺-dependentC_m responses were corrected for transient ΔC_m artifacts (as explained in text) by subtracting the response to a 5 msec depolarization aligned with the pulse. C. Integrated Ca²⁺ currents (open circles; n = 8) and corresponding immediate (filled circles;n = 8) and slow (filled squares; n = 6)C_m responses plotted against command potential at which they were evoked.

Fig. 4.

Activation of κ-opioid receptors attenuates immediate and slow C_m responses evoked by steps inducing smaller Ca²⁺ influx.C_m increases (A₁,A₂, B₁,B₂) and corresponding Ca²⁺currents (A₃, B₃) recorded in response to 200 msec depolarizations to +10 mV (A) and +30 mV (B) from a holding potential of −90 mV. Administration of U69,593 appreciably reduced Ca²⁺ influx (32.1%, from 18.53 to 12.58 pC) evoked by depolarization to +10 mV but had little effect on the corresponding immediate C_m response (9% reduction, from 49 to 44 fF, indicated by dotted lines). Depolarization to +30 mV induced much smaller Ca²⁺influx (9.1 pC) that was attenuated further by activation of κ-opioid receptors (to 6.23 pC, representing 31.5% blockade). Under this condition of reduced driving force for Ca²⁺ influx, inhibition of the immediate C_m response by the κ-opioid agonist was markedly increased (47%, from 36 to 19 fF). Opioid administration also reduced the slowly increasingC_m component recorded in this nerve ending, and this effect of U69,593 was greater on the response measured at +30 mV (33.9%, from 118 to 78 fF) compared with that recorded at +10 mV (17.7%, from 192 to 158 fF). C, Bar graph summarizes the effects of U69,593 on the immediate step and slowly increasing components of C_m responses. Bars represent the mean ± SEM of the normalized C_mresponses pooled from κ-opioid-responsive terminals (number shown in numerator, out of the total number of terminals examined, shown in denominator) for immediate and slowC_m responses evoked at command potentials of +10 or +30 mV, expressed as a percent of the corresponding predrug control values. Reduction of depolarization-evoked Ca²⁺ influx to submaximal levels resulted in an increased probability for modulation of the immediateC_m response by κ-opioid receptor activation from 32% (13 of 41) at +10 mV to 100% (6 of 6) at +30 mV, without significantly (p = 0.19,NS indicates not significant) altering the magnitude of the inhibitory effect of U69,593 on these initial exocytotic events. In contrast, the inhibitory effect of U69,593 on the slowC_m responses was significantly increased under conditions in which the driving force for Ca²⁺influx and the net amount of depolarization-evoked Ca²⁺ influx was reduced (asteriskindicates p < 0.001).

Fig. 5.

Activation of κ-opioid receptors attenuates depolarization-evoked exocytotic C_mresponses in neurohypophysial nerve endings in nor-BNI-sensitive manner. C_m increases (A₁, B₁) and corresponding Ca²⁺ currents (A₂, B₂) recorded in response to 200 msec depolarizations to +10 mV from a holding potential of −90 mV in control solution (A) and in the presence of the selective κ-opioid antagonist nor-BNI (B). Administration of U69,593 reduced the evoked Ca²⁺ current and accompanying slowly increasingC_m response, and these effects were prevented after blockade of κ-opioid receptors by nor-BNI (1 μm).

Fig. 6.

Comparison of effects of U69,593 and dynorphin A demonstrates the similar efficacy of the two κ-opioid agonists in inhibiting Ca²⁺ currents and slowC_m responses. C_mincreases (A₁, B₁) and corresponding Ca²⁺ currents (A₂, B₂) recorded in the same nerve terminal in response to 200 msec depolarizations to +10 mV from a holding potential of −90 mV in control solution and in the presence of U69,593 (A) and dynorphin A (B). Administration of U69,593 reduced the evoked Ca²⁺ current and accompanying slowly increasingC_m response to a degree similar to that seen with dynorphin A.