Development of cross-tolerance to 5-hydroxytryptamine in organotypic cultures of mouse spinal cord-ganglia during chronic exposure to morphine

Exposure of organotypic explants of mouse spinal cord with attached dorsal root ganglia (DRGs) to low concentrations (～10nM) of 5-hydroxytryptamine (5-HT) markedly depressed sensory-evoked dorsal-horn network responses, resembling the acute effects of opioids in these cultures. Attenuation of cord responses by 5-HT was not prevented by exposure to the 5-HT antagonists, methysergide and cyproheptadiene, nor to the opiate antagonist, naloxone. Explants that had become tolerant to morphine after chronic exposure (1 μM) for > 2 days often developed cross-tolerance to 5-HT. Acute exposure of morphine-tolerant explants to naloxone (1 μM) further attenuated the effects of 5-HT so that the minimum depressant levels of 5-HT were often increased up to 30-fold. Increasing the extra-cellular Ca⁺⁺ concentration (to 5 mM) and/or introduction of 4-aminopyridine markedly antagonized the depressant effects of 5-HT on DRG-evoked cord responses, so that 5-HT levels comparable to those used on morphine-tolerant explants were required to depress naive explants. These depressant effects of 5-HT on cord-DRG explants are consonant with antinociceptive actions of 5-HT administered to dorsal cord $\text{in situ}$. Our data suggest that 5-HT may block neuronal components of dorsal horn networks at similar regions to those that are depressed by opiates, e.g. presynaptic DRG nerve terminals where abundant opiate receptors are located. The marked attenuation of the depressant effects of both 5-HT and opiates on cord-DRG explants by high Ca⁺⁺ raises the possibility that cross-tolerance to 5-HT in morphine-tolerant explants may result from the same neuronal alterations that render dorsal-horn networks tolerant to opiates. Furthermore, the increased degree of cross-tolerance to 5-HT after acute introduction of naloxone in morphine-tolerant cultures may be an expression of opiate dependence.     

Development of tolerance to opiates and opioid peptides in organotypic cultures of mouse spinal cord

Following chronic exposure of organotypic explants of mouse spinal cord with attached dorsal root ganglia (DRG) to low levels of morphine (1 μM) for 2–3 days (at 35°C), the initial opiate-depressant effects on sensory-evoked dorsal-horn network responses disappeared and characteristic dorsal cord responses could then be evoked by DRG stimuli in the presence of morphine — even after acute increases in concentration (up to 100-fold). Similar tolerance developed after chronic exposure of cord-DRG explants to low concentrations (10 nM) of an enkephalin analog (Sandoz FK 33-824). The latter cultures showed cross-tolerance to met-enkephalin and opiates; dorsal cord responses could still be evoked even after acute exposure to high levels of morphine. Morphine-tolerant cultures also showed cross-tolerance to met-enkephalin and to the Sandoz opioid peptide (FK 33-824). The tolerant state did not develop if the cultures were incubated at lower temperature, ca. 20°C, during exposure to 1 μM morphine for as long as 7 days. The data indicate that a temperature-dependent metabolic change occurs in these neurons after chronic exposure to morphine at 35°C leading to a sustained decrease in sensitivity to opiate-depressant effects. Enhanced dorsal-horn responses in tolerant cultures suggested development of “dependence” as well as tolerance to opiates in these isolated cord-DRG tissues.     

A presynaptic locus of the action of Met-enkephalin demonstrated in mouse spinal cord cultures

Monosynaptic excitatory post-synaptic potentials (EPSPs) evoked in spinal cord (SC) neurons by stimulation of dorsal root ganglion (DRG) neurons in cell cultures were reduced by perfusion application of the opiate peptide, Met-enkephalin (2-4 microM). In about 2/3 of cases examined, EPSPs evoked by stimulation of spinal cord cells were also reduced by Met-enkephalin. The effects were antagonized by concomitant perfusion with naloxone (1-2 microM) and recovered when perfusion with Met-enkephalin was stopped. Statistical analysis of synaptic responses indicated that the reduction of EPSP amplitude was due, at least to a major extent, to a decrease in presynaptic transmitter release.     

Reversal of the antinociceptive effects of centrally-administered morphine by the benzodiazepine receptor antagonist Ro 15-1788

The effects of the benzodiazepine receptor antagonist, Ro 15-1788, were examined on analgesia induced by morphine after central (intracerebroventricular, i.c.v., or intrathecal, i.t.) and systemic administration. Analgesia was assessed in squirrel monkeys trained to respond under an electric shock tiltration procedure and in mice using the radiant heat tail-flick test. Central and systemic administration of morphine produced antinociceptive effects that were antagonized by 0.1 mg/kg of naloxone in both species. Ro 15-1788 antagonized the effects of morphine after central (i.c.v. or i.t.) administration but did not alter the effects of morphine given by the systemic route. This novel interaction suggests that Ro 15-1788 may be useful in pharmacologically separating neural substrates subserving opiate analgesia.     

Ionic mechanisms of action of GABA on dorsal and ventral root myelinated fibers: effects of K+ channel blockers

Excitability changes evoked by the inhibitory neurotransmitter, GABA (gamma-aminobutyric acid) in myelinated axons of dorsal and ventral roots of the isolated bullfrog sciatic nerve were compared in the absence and presence of K+ channel blockers. Half-maximal A-fiber responses to a 0.5-Hz stimulation of the whole nerve were recorded from individual roots. Direct applications of Ringer with raised K+ levels to the site of stimulation caused increases in excitability of both dorsal and ventral root fibers, which resembled those evoked in the ventral root by the GABA agonist THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]ol). The increases in dorsal root fiber responses produced by GABA were depressed by tetraethylammonium (TEA) (3 mM), 4-aminopyridine (4-AP) (50 microM), Cs (2 mM), and Ba (1 mM). Ventral root fibers were less consistently affected. The early component of GABA-evoked excitability increases was depressed by 4-AP, Cs, and Ba, but greatly augmented by TEA. THIP-evoked changes in the excitability of the dorsal and ventral root fibers were, respectively, depressed and enhanced by TEA. The augmenting effect of TEA on the early component of GABA agonist effects on the ventral root fibers is attributed to their high resting K+ conductance and the presence of a slowly inactivating, fast K+ current (If1). The depressant effects of K+ channel blockade on depolarizing components of agonist-evoked changes in dorsal and ventral root responses indicate interference with release and (or) sensitivity to K+ and a possible contribution from a mechanism involving voltage-dependent delayed rectifier K+ currents.     

Differential Sensitivity to Blockade by 4-Aminopyridine of Presynaptic Receptors Regulating [3H]Acetylcholine Release from Rat Hippocampus

The inhibitory effect of an adenosine analogue, R-N6-phenylisopropyl adenosine (R-PIA), of the cholinergic agonist carbachol, and of morphine on 3H efflux from [3H]choline-labeled field-stimulated rat hippocampal slices was compared with that produced by two inhibitors of N- and L-type Ca2+ channels, omega-conotoxin (CgTx; conotoxin GVIA) and cadmium chloride. 4-Aminopyridine (4-AP) caused a dose-dependent increase in evoked transmitter release, with a maximal effect (an almost threefold increase) at 100 microM. 4-AP (100 microM) did not affect the actions of CgTx, cadmium chloride, and R-PIA but almost abolished the effect of carbachol and morphine. The present results indicate that presynaptic muscarinic and opiate receptors reduce acetylcholine release by a mechanism that is somewhat different from that used by adenosine A1 receptors. Furthermore, the results indicate that presynaptic A1 receptors on hippocampal cholinergic neurons do not primarily regulate 4-AP-dependent potassium channels, but that they might act directly on a Ca2+ conductance.     

Interactions of Schwann cells with neurites and with other Schwann cells involve the calcium-dependent adhesion molecule, N-cadherin.

During embryogenesis, Schwann cells interact with axons and other Schwann cells, as they migrate, ensheath axons, and participate in organizing peripheral nervous tissues. The experiments reported here indicate that the calcium-dependent molecule, N-cadherin, mediates adhesion of Schwann cells to neurites and to other Schwann cells. Cell cultures from chick dorsal root ganglia and sciatic nerves were maintained in media containing either 2 mM Ca++ or 0.2 mM Ca++, a concentration that inactivates calcium-dependent cadherins. When the leading lamellae of Schwann cells encountered migrating growth cones in medium with 2 mM Ca++, they usually remained extended, and the growth cones often advanced onto the Schwann cell upper surface. In the low Ca++ medium, the frequency of withdrawal of the Schwann cell lamella after contact with a growth cone was much greater, and withdrawal was the most common reaction to growth cone contact in medium with 2 mM Ca++ and anti-N-cadherin. Similarly, when motile leading margins of two Schwann cells touched in normal Ca++ medium, they often formed stable areas of contact. N-cadherin and vinculin were co-concentrated at these contact sites between Schwann cells. However, in low Ca++ medium or in the presence of anti-N-cadherin, interacting Schwann cells usually pulled away from each other in a behavior reminiscent of contact inhibition between fibroblasts. In cultures of dissociated cells in normal media, Schwann cells frequently were aligned along neurites, and ultrastructural examination showed extensive close apposition between plasma membranes of neurites and Schwann cells. When dorsal root ganglia explants were cultured with normal Ca++, Schwann cells migrated away from the explants in close association with extending neurites. All these interactions were disrupted in media with 0.2 mM Ca++. Alignment of Schwann cells along neurites was infrequent, as were extended close apposition between axonal and Schwann cell plasma membranes. Finally, migration of Schwann cells from ganglionic explants was reduced by disruption of adhesive contact with neurites. The addition of antibodies against N-cadherin to medium with normal Ca++ levels had similar effects as lowering the Ca++ concentration, but antibodies against the neuronal adhesive molecule, L1, had no effects on interactions between Schwann cells and neurites.     

Effects of deprolorphin, a casomorphin analog, on hippocampal CA1 field potentials in vitro

The effects of infusion of low concentrations of the synthetic opioid peptide D-Pro4-beta-casomorphin-5(deprolorphin) on electrical field responses in the in vitro hippocampal slice preparation of mice were investigated. Deprolorphin (0.01-10 microM) causes a large enhancement of the population spike (PS) and appearance of additional spikes of CA1 pyramidal cells to Schaffer-commissural stimulation, which were partially antagonized by the opiate receptor antagonist naloxone. It is likely that this analgesic peptide in the hippocampus acts through mu-receptors and neuronal mechanisms already described for morphine and enkephalin analogs.     

Opposite Effects of δ and μ Opioid Receptor Agonists on the In Vitro Release of Substance P-Like Material from the Rat Spinal Cord

Superfusion of slices from the dorsal half of the lumbar enlargement of rat spinal cord with Krebs-Henseleit medium supplemented with 30 microM bacitracin allowed the collection of substance P-like immunoreactive material (SPLI), which was released at a rate of approximately 10 pg/4 min. Tissue depolarization by an excess of K+ (30-60 mM) or veratridine (50 microM) induced a marked increase in SPLI outflow, provided that Ca2+ was present in the superfusing fluid. K+- or veratridine-induced SPLI overflow could be modulated in opposite directions by mu and delta opioid receptor agonists. Thus, the two preferential mu agonists Tyr-D-Ala-Gly-MePhe-Gly-ol (DAGO; 10 microM) and Tyr-D-Ala-Gly-MePhe-Met(O)5-OH (FK-33824; 0.1 microM) enhanced SPLI overflow from depolarized tissues, whereas the selective delta agonists Tyr-D-Thr-Gly-Phe-Leu-Thr (deltakephalin; 3 microM) and [2-D-penicillamine, 5-D-penicillamine]enkephalin (50 microM) reduced it. The effect of DAGO was antagonized by a low concentration (1 microM) of naloxone but not by the selective delta antagonist ICI-154129 (50 microM). In contrast, the latter drug prevented the inhibitory influence of delta agonists on K+-induced SPLI release. Complementary experiments with morphine (10 microM) and [2-D-alanine, 5-D-leucine]enkephalinamide (3 microM), in combination with 1 microM naloxone or 50 microM ICI-154129 for the selective blockade of mu or delta receptors, respectively, confirmed that the stimulation of mu receptors increased, whereas the stimulation of delta receptors reduced, SPLI overflow. The results suggest that, at the spinal level, and antinociceptive action of delta but not mu agonists might involve a presynaptic inhibition of substance P-containing primary afferent fibers.     

Pharmacological Characterization of Morphine-Induced In Vivo Release of Cholecystokinin in Rat Dorsal Horn

Previous studies indicate that an increased release of cholecystokinin (CCK) in response to morphine administration may counteract opioid-induced analgesia at the spinal level. In the present study we used in vivo microdialysis to demonstrate that systemic administration of antinociceptive doses of morphine (1-5 mg/kg, s.c.) induces a dose-dependent and naloxone-reversible release of CCK-like immunoreactivity (CCK-LI) in the dorsal horn of the spinal cord. A similar response could also be observed following perfusion of the dialysis probe for 60 min with 100 microM but not with 1 microM morphine. The CCK-LI release induced by morphine (5 mg/kg, s.c.) was found to be calcium-dependent and tetrodotoxin-sensitive (1 microM in the perfusion medium). Topical application of either the L-type calcium channel blocker verapamil (50 microg) or the N-type calcium channel blocker omega-conotoxin GVIA (0.4 microg) onto the dorsal spinal cord completely prevented the CCK-LI release induced by morphine (5 mg/kg, s.c.). Our data indicate that activation of L- and N-type calcium channels is of importance for morphine-induced CCK release, even though the precise site of action of morphine in the dorsal horn remains unclear. The present findings also suggest a mechanism for the potentiation of opioid analgesia by L- and N-type calcium channel blocking agents.     

强啡肽A和CCK—8对大鼠脊髓突触小体摄取^45Ca的影响

为了探讨血管紧张素Ⅱ(AⅡ)和八肽胆囊收缩素(CCK-8)这两种肽的抗阿片作用机理,本实验中观察了三种阿片类物质(吗啡、强啡肽和 DPDPE)和两种抗阿片物质(AⅡ和 CCK-8)对大鼠脊髓突触小体摄取~(45) Ca 的影响。结果表明:(1)在脊髓腹柱突触小体上,10nmol/L—1μmol/L 的吗啡、强啡肽 A(Dyn A)和 DPDPE 对~(45)Ca 摄取均有较弱的抑制作用;(2)CCK-8在浓度高达lμmol/L 时对~(45)Ca 摄取有较弱的抑制作用;(3)AⅡ在浓度高达lμmol/L时也不影响腹柱突触小体摄取~(45)Ca;(4)在背柱的突触小体制备中,上述阿片物质中 Dyn A 对~(45)Ca 摄取有较强的抑制作用,并被 k 受体阻断剂 nor-BNI 所阻断。10和100nmol/L 的 CCK-8能翻转lμmol/L Dyn A 对~(45)Ca 摄取的抑制作用;(5)A Ⅱ不能翻转Dyn A 的抑制作用。以上结果提示,CCK-8阻断 Dyn A 抑制脊髓背柱突触小体摄取 Ca~(2+)的作用可能是其行为学中抗阿片作用的机理之一。AⅡ对脊髓 Ca~(2+)摄取和 Dyn A 抑制脊髓 Ca~(2+)摄取的作用皆无影响,与行为学中观察到的 AⅡ在脊髓内不能对抗阿片镇痛的现象一致,进一步说明 CCK-8和AⅡ拮抗阿片类物质对神经末梢 Ca~(2+)摄取的影响可能是其抗阿片作用的重要机理之一。     

Voltage-dependent K+ channels regulate the duration of reactive hyperemia in the canine coronary circulation

We previously demonstrated a role for voltage-dependent K(+) (K(V)) channels in coronary vasodilation elicited by myocardial metabolism and exogenous H(2)O(2), as responses were attenuated by the K(V) channel blocker 4-aminopyridine (4-AP). Here we tested the hypothesis that K(V) channels participate in coronary reactive hyperemia and examined the role of K(V) channels in responses to nitric oxide (NO) and adenosine, two putative mediators. Reactive hyperemia (30-s occlusion) was measured in open-chest dogs before and during 4-AP treatment [intracoronary (ic), plasma concentration 0.3 mM]. 4-AP reduced baseline flow 34 +/- 5% and inhibited hyperemic volume 32 +/- 5%. Administration of 8-phenyltheophylline (8-PT; 0.3 mM ic or 5 mg/kg iv) or N(G)-nitro-L-arginine methyl ester (L-NAME; 1 mg/min ic) inhibited early and late portions of hyperemic flow, supporting roles for adenosine and NO. 4-AP further inhibited hyperemia in the presence of 8-PT or L-NAME. Adenosine-induced blood flow responses were attenuated by 4-AP (52 +/- 6% block at 9 microg/min). Dilation of arterioles to adenosine was attenuated by 0.3 mM 4-AP and 1 microM correolide, a selective K(V)1 antagonist (76 +/- 7% and 47 +/- 2% block, respectively, at 1 microM). Dilation in response to sodium nitroprusside, an NO donor, was attenuated by 4-AP in vivo (41 +/- 6% block at 10 microg/min) and by correolide in vitro (29 +/- 4% block at 1 microM). K(V) current in smooth muscle cells was inhibited by 4-AP (IC(50) 1.1 +/- 0.1 mM) and virtually eliminated by correolide. Expression of mRNA for K(V)1 family members was detected in coronary arteries. Our data indicate that K(V) channels play an important role in regulating resting coronary blood flow, determining duration of reactive hyperemia, and mediating adenosine- and NO-induced vasodilation.     

Voltage-dependent calcium and calcium-activated potassium currents of a molluscan photoreceptor

Two-microelectrode voltage clamp studies were performed on the somata of Hermissenda Type B photoreceptors that had been isolated by axotomy from all synaptic interaction as well as any impulse-generating (i.e., active) membrane. In the presence of 2-10 mM 4-aminopyridine (4-AP) and 100 mM tetraethylammonium ion (TEA), which eliminated two previously described voltage-dependent potassium currents (IA and the delayed rectifier), a voltage-dependent outward current was apparent in the steady state responses to command voltage steps more positive than -40 mV (absolute). This current increased with increasing external Ca++. The magnitude of the outward current decreased and an inward current became apparent following EGTA injection. Substitution of external Ba++ for Ca++ also made the inward current more apparent. This inward current, which was almost eliminated after being exposed for approximately 5 min to a solution in which external Ca++ was replaced with Cd++, was maximally activated at approximately 0 mV. Elevation of external potassium allowed the calcium (ICa++) and calcium-dependent K+ (IC) currents to be substantially separated. Command pulses to 0 mV elicited maximal ICa++ but no IC because no K+ currents flowed at their new reversal potential (0 mV) in 300 mM K+. At a holding potential of -60 mV, which was now more negative than the potassium equilibrium potential, EK+, in 300 mM K+, IC appeared as an inward tail current after positive command steps. The voltage dependence of ICa++ was demonstrated with positive steps in 100 mM Ba++, 4-AP, and TEA. Other data indicated that in 10 mM Ca++, IC underwent pronounced and prolonged inactivation whereas ICa++ did not. When the photoreceptor was stimulated with a light step (with the membrane potential held at -60 mV), there was also a prolonged inactivation of IC. In elevated external Ca++, ICa++ also showed similar inactivation. These data suggest that IC may undergo prolonged inactivation due to a direct effect of elevated intracellular Ca++, as was previously shown for a voltage-dependent potassium current, IA. These results are discussed in relation to the production of training-induced changes of membrane currents on retention days of associative learning.     

A comparison of the analgesic and behavioral effects of [D-Ala2] Met-enkepha-linamide and morphine in the mesencephalic reticular formation of rats.

[D-Ala²]Met-enkephalinamide (DALA) injected intracerebrally (IC) at low doses into specific sites of the mesencephalic reticular formation (MRF), produced a profound, long-lasting analgesia that was blocked by naloxone, a specific opiate antagonist. Morphine was only half as potent as DALA because morphine, injected IC at similar sites in the MRF, yielded a comparable analgesia only when injected at twice the dose. The analgesic effects of morphine were also antagonized by naloxene. Both DALA and morphine produced specific behavioral effects. Naloxone blocked the behavioral effects of DALA, but not those produced by morphine.     

Hyperpolarization contributes to vascular hyporeactivity in rats with lipopolysaccharide-induced endotoxic shock

We have examined the role of membrane hyperpolarization in mediating vascular hyporeactivity induced by bacterial lipopolysaccharide (LPS) in endothelial-denuded strips of rat thoracic aorta ex vivo. The injection of rats with LPS caused a significant fall of blood pressure and a severe vascular hyporeactivity to norepinephrine. The membrane potential recording showed that endotoxemia caused a hyperpolarization when compared to the control. This hyperpolarization was fully restored by methylene blue (MB; 10 microM) and partially reversed by Nomega-nitro-L-arginine methyl ester (L-NAME; 0.3 mM), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 1 microM), tetraethylammonium (TEA; 10 mM), charybdotoxin (CTX; 0.1 microM), or glibenclamide (GB; 10 microM), however, this hyperpolarization was not significantly affected by apamin (0.1 microM), 4-aminopyridine (4-AP; 1 mM), or Ba2+ (50 microM). In addition, the basal tension of the tissues obtained from endotoxemic rats was enhanced by the following order: MB > or = ODQ > TEA > or = L-NAME > or = CTX > GB; whereas apamin, 4-AP or Ba2+ had no significant effects on these tissues. In contrast, none of these inhibitors had significant effects on the membrane potential or the basal tension in control tissues. Our electrophysiological results further confirmed previous studies showing that in addition to nitric oxide, the large conductance Ca2+-activated K+-channels and ATP-sensitive K+-channels are, most likely, responsible for endotoxin-mediated hyporeactivity to vasoconstrictor agents in vascular smooth muscle.     

Voltage-dependent ebselen and diorganochalcogenides inhibition of 45Ca2+ influx into brain synaptosomes

By mediating the Ca(2+) influx, Ca(2+) channels play a central role in neurotransmission. Chemical agents that potentially interfere with Ca(2+) homeostasis are potential toxic agents. In the present investigation, changes in Ca(2+) influx into synaptosomes by organic forms of selenium and tellurium were examined under nondepolarizing and depolarizing conditions induced by high KCl concentration (135 mM) or by 4-aminopyridine (4-AP). Under nondepolarizing conditions, ebselen (400 micro M) increased Ca(2+) influx; diphenyl ditelluride (40-400 micro M) decreased Ca(2+) in all concentrations tested; and diphenyl diselenide decreased Ca(2+) influx at 40 and 100 micro M, but had no effect at 400 micro M. In the presence of KCl as depolarizing agent, ebselen and diphenyl ditelluride decreased Ca(2+) influx in a linear fashion. In contrast, diphenyl diselenide did not modify Ca(2+) influx into isolated nerve terminals. In the presence of 4-AP (3 mM) as depolarizing agent, ebselen (400 micro M) caused a significant increase, whereas diphenyl diselenide and diphenyl ditelluride inhibited Ca(2+) influx into synaptosomes. The results can be explained by the fact that the mechanism through which 4-AP and high K(+) induced elevation of intracellular Ca(2+) is not exactly coincident. The mechanism by which diphenyl ditelluride and ebselen interact with Ca(2+) channel is unknown, but may be related to reactivity with critical sulfhydryl groups in the protein complex. The results of the present study indicate that the effects of organochalcogenides were rather complex depending on the condition and the depolarizing agent used.     

Characterization of the Participation of Sodium Channels on the Rise in Na+ Induced by 4-Aminopyridine (4-AP) in Synaptosomes

The participation of voltage-sensitive Na+ channels (VSSC) on the changes on internal (i) Na+, K+, Ca2+, and on DA, Glu, and GABA release caused by different concentrations of 4-AP was investigated in striatum synaptosomes. TTX, which abolished the increase in Na(i) (as determined with SBFI), induced by 0.1 mM 4-AP only inhibited by 30% the rise in Na(i) induced by 1 mM 4-AP. One millimolar 4-AP markedly decreased the fluorescence of the K+ indicator dye PBFI but 0.1 mM 4-AP did not. Like 1 mM 4-AP, ouabain decreased PBFI fluorescence and increased a considerable fraction of Na(i) in a TTX-insensitive manner. In contrast with the different TTX sensitivity of the rise in Na(i) induced by 0.1 and 1 mM 4-AP, the rise in Ca(i) (as determined with fura-2) induced by the two concentrations of 4-AP was markedly inhibited by TTX, as well as by omega-agatoxin in combination with omega-conotoxin GVIA, indicating that only the TTX-sensitive fraction of the rise in Na(i) induced by 4-AP is linked with the activation of presynaptic Ca2+ channels. It is concluded that the TTX-sensitive fraction of neurotransmitter release evoked by 4-AP is released by exocytosis, and the TTX insensitive fraction involves reversal of the neurotransmitters transporters. This contrasts with the exocytosis evoked by high K+ that is unchanged by TTX and with the neurotransmitter-transporter-mediated release evoked by veratridine, which is highly TTX sensitive and does not require activation of Ca2+ channels.     

A novel kappa agonist inhibits [3H]nimodipine binding to a Ca++ channel receptor protein in rat brain

The novel kappa agonist U50-488H in vitro produced a concentration-dependent decrease (0.25-25 microM) in [3H]nimodipine binding in neuronal P2 fractions [corrected] from rat brain cortex. Kinetic analysis indicates the decrease in binding results from a reduced Bmax with no change in affinity (Kd). The kappa antagonist, MR2266, blocked the decrease in [3H]nimodipine binding to membrane fractions. At equimolar concentrations (25 microM), morphine in vitro had no effect on [3H]nimodipine binding, while U50-488H demonstrated potent inhibition. Further kinetic analysis indicates that the IC50 for U50-488H is 0.5-0.7 microM with a KI by a Dixon plot of 1.5-1.7 microM [corrected]. These results suggest that kappa opiate receptors may be coupled to dihydropyridine receptors and as a result modulate Ca++ entry and neurotransmitter release in brain neurons.     

Ammonium ions mobilize calcium from an internal pool which is insensitive to TRH and ionomycin in bovine anterior pituitary cells

The effects of NH4Cl on cytoplasmic free calcium concentration ([Ca2+]i) and pH (pHi) in single bovine anterior pituitary cells were determined using fluorescence imaging microscopy. Addition of NH4Cl (10-40 mM) in the presence of 1 mM extracellular calcium ([Ca2+]e) increased [Ca2+]i to a peak which then fell to a sustained plateau, returning to resting levels upon removal of NH4Cl. In medium containing 0.1 microM [Ca2+]e, or in 1 mM [Ca2+]e medium containing 0.1 microM nitrendipine, the plateau was absent leaving only a transient [Ca2+]i spike. NH4Cl also increased pHi and this, like the [Ca2+]i plateau, remained elevated during the continued presence of NH4Cl. In medium containing only 0.1 microM [Ca2+]e, to preclude refilling of internal stores by entry of external calcium, repeated exposures to NH4Cl induced repeated [Ca2+]i transients. In contrast, only the initial exposure to thyrotropin releasing hormone (TRH; 20-500 nM) caused a [Ca2+]i rise but, after an additional exposure to NH4CI, TRH responses re-emerged in some cells. Pre-treatment with the calcium ionophore ionomycin abolished the rise caused by TRH, but neither TRH nor ionomycin pretreatment affected the response to NH4Cl. Neither acetate removal nor methylamine increased [Ca2+]i in medium containing 0.1 microM [Ca2+]e, although in both cases pHi increased. We conclude that in bovine anterior pituitary cells NH4Cl raises [Ca2+]i by two independent pathways, increasing net calcium entry and mobilizing Ca2+ from a TRH-insensitive calcium store.     

Inhibition of the acetylcholine-induced relaxation of canine isolated basilar artery by potassium-conductance blockers

Canine basilar artery rings precontracted with 5-hydroxytryptamine (0.1-0.5 microM) relaxed in the presence of acetylcholine (25-100 microM), sodium nitroprusside (0.1 microM), or stimulation of the electrogenic sodium pump by restoration of extracellular K+ (4.5 mM) after K(+)-deprivation. Acetylcholine-induced relaxation is believed to be caused by the release of endothelium-derived relaxing factor (EDRF) and is prevented by mechanical removal of the endothelium, while relaxations induced by sodium nitroprusside or restarting of the sodium pump are endothelium-independent. Acetylcholine-induced relaxation was selectively blocked by pretreatment of the tissue with the nonselective K+ conductance inhibitors, 4-aminopyridine (4-AP, 3 mM), Ba2+ (1 mM), and tetraethylammonium (20 mM), 4-AP also blocked ACh-mediated relaxation in muscles contracted with elevated external K+. Relaxation of 5-hydroxytryptamine-induced contraction by sodium nitroprusside, or by addition of K+ to K(+)-deprived muscle, was not affected by 4-AP. Relaxation of basilar artery with acidified sodium nitrite solution (containing nitric oxide) was reduced by 4-AP. These results suggest that 4-AP and possibly Ba2+ inhibit acetylcholine-induced endothelium-dependent relaxation by inhibition of the action of EDRF on the smooth muscle rather than through inhibition of release of EDRF. The increase in K+ conductance involved in acetylcholine-induced relaxation is not due to ATP-inhibited K+ channels, as it is not blocked by glyburide (10(-6) M). Endothelium-derived relaxant factor(s) may relax smooth muscle by mode(s) of action different from that of sodium nitroprusside or by hyperpolarization due to the electrogenic sodium pumping.(ABSTRACT TRUNCATED AT 250 WORDS)