Endogenous enkephalins and ␦ opiates affect sensory function and pain sensation by inhibiting synaptic transmission in sensory circuits via delta opioid receptors (DORs). DORs have long been suspected of mediating these effects by modulating voltage-dependent Ca 2ϩ entry in primary sensory neurons. However, not only has this hypothesis never been validated in these cells, but in fact several previous studies have only turned up negative results. By using whole-cell current recordings, we show that the ␦ enkephalin analog [D-Ala 2 , D-Leu 5 ]-enkephalin (DADLE) inhibits, via DORs, L-, N-, P-, and Q-high voltageactivated Ca 2ϩ channel currents in cultured rat dorsal root ganglion (DRG) neurons. The percentage of responding cells was remarkably high (75%) within a novel subpopulation of substance P-containing neurons compared with the other cells (18-35%). DADLE (1 M) inhibited 32% of the total barium current through calcium channels (I Ba ). A ␦ (naltrindole, 1 M), but not a (␤-funaltrexamine, 5 M), antagonist prevented the DADLE response, whereas a DOR-2 subtype (deltorphin-II, 100 nM), but not a DOR-1 (DPDPE, 1 M), agonist mimicked the response. L-, N-, P-, and Q-type currents contributed, on average, 18, 48, 14, and 16% to the total I Ba and 19, 50, 26, and 20% to the DADLE-sensitive current, respectively. The druginsensitive R-type current component was not affected by the agonist. This work represents the first demonstration that DORs modulate Ca 2ϩ entry in sensory neurons and suggests that ␦ opioids could affect diverse Ca 2ϩ -dependent processes linked to Ca 2ϩ influx through different high-voltage-activated channel types.

The Shaw-type K+ channel Kv3.1 was stably transfected in human embryonic kidney cells. Voltage dependence of activation, K+ permeability, sensitivity to external tetraethylammonium, and unitary conductance were similar to Kv3.1 channels expressed transiently in Xenopus oocytes. Kv3.1 channels appear to be regulated because the protein kinase C activator phorbol 12,13-dibutyrate decreased Kv3.1 currents. Based on these results, we find that the stable expression of voltage-gated K+ channels in human embryonic kidney cells appears to be well suited for analysis of both biophysical and biochemical regulatory processes.

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The involvement of G proteins in the transduction mechanism of M current (Im) inhibition by extracellular ligands in bullfrog sympathetic neurons was examined using the hydrolysis resistant nucleotide analogues GTPgammaS and GDPbetaS. Im was recorded in large (40 - 60 microm) isolated neurons using the patch-clamp technique in the whole-cell configuration, as well as in neurons from the intact ganglion impaled with conventional microelectrodes. In whole-cell recordings Im could be recorded without significant loss for 1 h or more provided ATP was present in the patch pipette. Muscarine, D-Ala6-LHRH, substance P and UTP reversibly inhibited Im in isolated control neurons, with full and rapid recovery of the current following agonist washout. Dialysis of isolated neurons with various concentrations of GTPgammaS (1 - 100 microM) affected, in a dose-dependent manner, the recovery of Im after its inhibition by brief agonist application. With 50 microM GTPgammaS, Im inhibition became completely irreversible. Similarly, the reversibility of Im inhibition by muscarine was reduced or abolished by the iontophoretic injection of GTPgammaS through a second microelectrode into neurons of the intact ganglion. GTPgammaS by itself caused a slow, agonist-independent suppression of Im in dialysed neurons, thus mimicking agonist action. Dialysis of isolated neurons with GDPbetaS (100 - 500 microM) attenuated by half or more the magnitude of Im inhibition by agonist as compared to control neurons. In addition, GDPbetaS attenuated the response of a given neuron to muscarine and D-Ala6-LHRH, and caused slow increase of Im, as a function of dialysis time. Incubation (2 - 72 h, 4 - 36 degrees C) of isolated neurons or intact ganglions with activated pertussis toxin had no effect on the response to muscarine. Toxin injections to experimental animals were equally ineffective. In contrast to Im, the additional inward current with increase in conductance induced by muscarine and D-Ala6-LHRH reversed with agonist washout in GTPgammaS-dialysed neurons, although more slowly than in control neurons. The results in this study indicate that a G protein, possibly pertussis toxin-insensitive, provides a common coupling step linking muscarinic, substance P, D-Ala6-LHRH and UTP receptors to the inhibition of M current.

We characterized a subpopulation of dorsal root ganglion (DRG) sensory neurons that were previously identified as preferential targets of enkephalins. This group, termed P-neurons after their "pear" shape, sequentially required nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) for survival in vitro during different developmental stages. Embryonic P-neurons required NGF, but not bFGF. NGF continued to promote their survival, although less potently, up to postnatal day 2 (P2). Conversely, at P5, they needed bFGF but not NGF, with either factor having similar effects at P2. This trophic switch was unique to that DRG neuronal group. In addition, neither neurotrophin-3 (NT-3) nor brain-derived neurotrophic factor influenced their survival during embryonic and postnatal stages, respectively. The expression of NGF (Trk-A) and bFGF (flg) receptors paralleled the switch in trophic requirement. No single P-neuron appeared to coexpress both Trk-A and flg. In contrast, all of them coexpressed flg and substance P, providing a specific marker of these cells. Immunosuppression of bFGF in newborn animals greatly reduced their number, suggesting that the factor was required in vivo. bFGF was present in the DRG and spinal cord, as well as in skeletal muscle, the peripheral projection site of P-neurons, as revealed by tracer DiIC(18)3. The lack of requirement of NT-3 for survival and immunoreactivity for the neurofilament of 200 kDa distinguished them from muscle proprioceptors, suggesting that they are likely to be unmyelinated muscle fibers. Collectively, their properties indicate that P-neurons constitute a distinct subpopulation of sensory neurons for which the function may be modulated by enkephalins.

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We studied the action of bradykinin (BK) on ionic currents in fused pheochromocytoma PC 12 cells under voltage-clamp in whole-cell mode, and on intracellular calcium using fura-2. BK induced the development of an outward current associated with an increase in intracellular calcium, followed by inhibition of an M-like current. The outward current was blocked by (+)-tubocurarine, and prevented when the calcium chelator BAPTA or high concentrations of inositol 1,4,5-triphosphate were introduced into the cell, whereas the M-like current and its inhibition by BK remained unaffected.The protein kinase activator phorbol 12,13 dibutyrate partially reduced the M-current. M-current density did not substantially change after prolonged treatment with nerve growth factor.M-current; Ca 2 + activated potassium current; Ca 2 + imaging; Protein kinase C; Bradykinin; Muscarine; (PC 12 cell)