L-type Ca²âº channel activity determines modulation of GABA release by dopamine in the substantia nigra reticulata and the globus pallidus of the rat.

Modulation of L-type Ca²âº-channel function by dopamine is a major determinant of the rate of action potential firing by striatal medium spiny neurons. However, the role of these channels in modulating GABA release by nerve terminals in the basal ganglia is unknown. We found that depolarization-induced [³H]GABA release in both the substantia nigra reticulata and the external globus pallidus (GPe), was depressed by about 50% by either the selective L-channel dihydropyridine blocker nifedipine or the P/Q channel blocker ω-agatoxin TK. The effects of these blockers were additive and together eliminated about 90% of depolarization-induced [³H]GABA release. In addition, in the substantia nigra reticulata, dihydropyridines prevented both the stimulation of [³H]GABA release produced by dopamine D1 receptor activation and the inhibition caused by D4 receptor activation. In the GP nifedipine blocked the effects of D2 and A2(A) receptor coactivation as well as the effects of activating adenylyl cyclase with forskolin. ω-Agatoxin TK did not interfere with the action of these modulatory agents. The L-type Ca²âº-channel agonist BAYK 8644 stimulated GABA release in both substantia nigra reticulata and GP. Because dihydropyridine sensitivity is a key criterion to identify L-type Ca²âº-channel activity, our results imply that these channels are determinant of GABA release modulation by dopamine in striatonigral, striatopallidal and pallidonigral terminals.

Dopamine inhibits GABA transmission from the globus pallidus to the thalamic reticular nucleus via presynaptic D4 receptors.

The globus pallidus sends a significant GABAergic projection to the thalamic reticular nucleus. Because pallidal neurons express D4-dopamine receptors, we have explored their presence on pallidoreticular terminals by studying the effect of dopamine and D4-receptor agonists on the GABAergic transmission in the thalamic reticular nucleus. We made whole-cell recordings of inhibitory postsynaptic currents (IPSCs) and miniature inhibitory postsynaptic currents (mIPSCs) in the thalamic reticular neurons. Dopamine consistently reduced the IPSCs. The effect of dopamine was associated with paired-pulse facilitation, indicating a presynaptic location of the receptors. The effect of dopamine was also measured on the mIPSCs, reducing their frequency but not affecting their amplitude, which also suggests a presynaptic site of action. The selective D4-receptor agonist PD 168,077 also reduced the IPSCs, which was also associated with paired-pulse facilitation. In addition, this agonist reduced the frequency of the mIPSCs with no effect on their amplitude. The D4-receptor antagonist L-745,870 totally blocked the effect of the D4-receptor agonist, indicating the specificity of its effect. To verify the location of the receptors on the pallidal terminals, these were eliminated by injecting kainic acid into the globus pallidus. Kainic acid produced a drastic (80%) fall in the globus pallidus neuronal population. In this condition, the effect of the activation of D4 receptors both on the IPSCs and mIPSCs was prevented, thus indicating that the location of the receptors was on the pallidal terminals. Our results demonstrate that dopamine controls the activity of the thalamic reticular neurons by regulating the inhibitory input from the globus pallidus.

Substantia nigra compacta neurons that innervate the reticular thalamic nucleus in the rat also project to striatum or globus pallidus: implications for abnormal motor behavior.

The projections of the substantia nigra pars compacta (SNc) to the reticular thalamic nucleus (RTn) were assessed by measuring dopamine content and counting tyrosine hydroxylase positive (TH (+)) cells in rats with unilateral lesions induced by 6-hydroxydopamine (6-OHDA), and by using a fluorescent tract-tracing technique in rats without lesions. Injection of 6-OHDA in the RTn reduced dopamine content and the number of TH (+) cells in the SNc by about 50%. Branching of SNc was suggested by the finding that 6-OHDA deposited in the RTn significantly reduced dopamine in the striatum and globus pallidus. Moreover, injections of 6-OHDA into either the striatum or the globus pallidus significantly reduced dopamine content in the RTn. Fluorescent tracers injected into the RTn labeled TH (+) cells in the SNc. A high proportion of these TH (+) cells was double labeled when tracers were also injected into either the globus pallidus or striatum. Other experiments showed that systemic injection of apomorphine or methamphetamine induced turning behavior in rats with local deposits of 6-OHDA in either the RTn or the studied basal ganglia nuclei. The extensive dopaminergic branching suggests that the abnormal motor behavior of rats with 6-OHDA deposits in the RTn may be caused by dopaminergic denervation of more than one structure. The fact that lesion of a single dopaminergic neuron can reduce dopamine transmission in more than one structure is probably important in generating the manifestations of Parkinson's disease.

L-DOPA inhibits depolarization-induced [3H]GABA release in the dopamine-denervated globus pallidus of the rat: the effect is dopamine independent and mediated by D2-like receptors.

The effect of L-DOPA on [(3)H]GABA release in slices of globus pallidus from 6-OHDA-lesioned rats was studied. Release was evoked by high (15 mM) K(+). The lesion reduced dopamine content and dopamine synthesized from L-DOPA. The inhibition of DOPA decarboxylase blocked dopamine synthesis. Endogenous dopamine released by high K(+) inhibited [(3)H]GABA release in normal but not in lesioned slices. L-DOPA inhibited (IC(50) = 0.44 microM) evoked [(3)H]GABA release. The inhibition was via D2-like receptors but not mediated by dopamine. The turning behavior induced by L-DOPA methyl ester (25 mg/kg, i.p.) was not abolished by the DOPA decarboxylase inhibitor 3-hydroxybenzylhydrazine but in this condition it was abolished by sulpiride. Results suggest that L-DOPA acting as D2-like agonist inhibits GABA release in the rat globus pallidus and induces turning behavior in rats with unilateral lesions of the dopamine innervation. L-DOPA could control Parkinson's disease symptoms acting not only as dopamine precursor but also by itself.

Adenosine A1 receptors control dopamine D1-dependent [(3)H]GABA release in slices of substantia nigra pars reticulata and motor behavior in the rat.

Abnormalities in dopaminergic control of basal ganglia function play a key role in Parkinson's disease. Adenosine appears to modulate the dopaminergic control in striatum, where an inhibitory interaction between adenosine and dopamine receptors has been demonstrated. However the interaction has not been established in substantia nigra pars reticulata (SNr) where density of both receptors is high. Here we have explored the interaction between A1/D1 receptors in SNr. In SNr slices, SKF 38393, a selective D1 receptor agonist, produced a stimulation of depolarization-induced Ca(2+)-dependent [(3)H]GABA release that was inhibited by adenosine. The adenosine inhibition was abolished by 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a selective adenosine A1 receptor antagonist. DPCPX per se enhanced GABA release, indicating inhibition of the release by endogenous adenosine. When D1 receptors were blocked with SCH 23390 or the slices were depleted of dopamine, the effect of DPCPX was suppressed, showing that activation of dopamine receptors was necessary for the adenosine inhibition. In normal slices, 2-chloro-n(6)-cyclopentyladenosine (CCPA), a selective A1 agonist, inhibited GABA release, but the inhibition was prevented by the blockade of D1 receptors with SCH 23390. Superperfusion with 8-bromo-cAMP produced a stimulation of GABA release that was not blocked by CCPA: this finding indicates that the blockade of D1 effects caused by activation of A1 receptors is specific. To see if these actions on GABA release were correlated with changes in motor behavior we studied the effect of unilateral intranigral injections of modifiers of adenosine A1 and dopamine D1 receptors in rats challenged with systemic methamphetamine. Both the A1 agonist CCPA and the D1 antagonist SCH 23390 produced ipsilateral turning whereas the A1 antagonist DPCPX caused contralateral turning. These motor effects are consistent with the findings on GABA release. The results indicate the presence of an inhibitory A1/D1 receptor interaction in SNr. The inhibition exerted by A1 adenosine receptors on GABAergic striatonigral transmission would be due exclusively to blockade of the facilitation resulting from activation of D1 dopamine receptors. The data permit to better understand the action of adenosine antagonists in the treatment of Parkinson's disease.

Intrapallidal dopamine restores motor deficits induced by 6-hydroxydopamine in the rat.

To explore whether dopamine deficits in the globus pallidus have a role in generating the motor symptoms of Parkinson's disease, we examined the effects of selective intrapallidal administration of dopamine or its antagonists in rats unilaterally lesioned with 6-hydroxydopamine into the medial forebrain bundle. Either the turning behavior induced by apomorphine or the deficit in the performance of a skilled forelimb-reaching task was used as assay for drug action. Microinjection of either the D2 receptor antagonist, sulpiride, or the D1 receptor antagonist, SCH-23390, into the dopamine-denervated pallidum significantly reduced apomorphine induced turning. In animals trained to perform a skilled forelimb-reaching task, 6-OHDA lesions caused a marked motor deficit in the contralateral forelimb. Intrapallidal dopamine applied either intermittently or continuously, restored up to 50% of the motor performance. Continuous application promoted a motor recovery that outlasted dopamine administration. These results show that lack of dopamine in the GP plays an important role in generating the motor symptoms caused by lesion of dopaminergic pathways. Moreover, motor recovery was produced by selectively injecting dopamine into the globus pallidus.

Intrapallidal D2 dopamine receptors control globus pallidus neuron activity in the rat.

Because activation of D2 dopamine receptors inhibits gamma-aminobutyric acid (GABA) release from intrapallidal nerve terminals, we measured the effects of modifiers of dopamine D2 receptors on the firing rate of single neurons in the globus pallidus (GP) of the anesthetized rat. The predominant effect of intrapallidal administration of the selective D2 agonist quinpirole was an increase in the rate of spontaneous firing while the D2 blocker sulpiride caused a decrease. The spontaneous firing of GP neurons is inhibited by stimulation of the GABAergic striatopallidal projection. We therefore measured the effects of modifiers of D2 receptors on striatal inhibition of GP neurons and found that intrapallidal quinpirole blocked the inhibitory effects of striatal stimulation while sulpiride enhanced them. These experiments show that both the spontaneous rate of firing of pallidal neurons and its modification by striatopallidal inputs is controlled by intrapallidal dopamine D2 receptors. In addition, taken together with other findings in the literature, our results suggest that activation of dopamine D2 receptors within the globus pallidus leads to inhibition of GABA release from presynaptic terminals.

Forskolin increases apical sodium conductance in cultured toad kidney cells (A6) by stimulating membrane insertion.

The role of membrane traffic in the stimulation of apical Na+ permeability caused by increases in cytoplasmic cyclic AMP was assessed by measuring the effects of forskolin on transepithelial capacitance (CT), transepithelial conductance (GT), and short-circuit current (Isc) in A6 cultured toad kidney cells. Apical water permeability was probed by recording cell volume changes after reducing the osmolality of the apical bath. We found that forskolin does not increase the osmotic water permeability of the apical membrane of A6 cells, and thus does not stimulate the insertion of water channels. Comparison of the effects of forskolin and insulin on Na+ transport demonstrated that both agents produce reversible increases in CT, GT and Isc. GT and CT increased proportionally during the rising phase of the insulin response. However, a non-linear relationship between both parameters was recorded when forskolin was given in NaCl Ringer's solution. The relationship between CT and GT became linear after the effects of forskolin on Cl- conductances were eliminated by substituting Cl- by an impermeant anion. In contrast, in Cl--containing Na+-free solutions, the non-linearity became more pronounced. Successive additions of insulin and forskolin caused additive increases in CT. Because increases in CT and Na+ transport occurred in the absence of stimulation of water permeability and increases of CT and GT were directly proportional when Na+ was the major permeating ion across the apical membrane, we suggest that the increase in apical Na+ permeability in the presence of either forskolin or insulin is due to the insertion of channels residing in intracellular pools. In contrast, the increased Cl- permeability caused by forskolin may be related to the activation of channels already present in the membrane.

Insulin effects on ouabain binding in A6 renal cells.

The effects of insulin on the Na+-K+-ATPase pump of the basolateral membrane of tight epithelia were evaluated by measuring transepithelial transport and [3H]ouabain binding in cultured A6 kidney cells. [3H]Ouabain binding in epithelia incubated in either K+-containing or K+-free solutions was measured. Insulin induced increases in transepithelial sodium transport, as measured by the short-circuit current (Isc), and in the initial rate of [3H]ouabain binding determined when the preparation was bathed in K+-containing solutions. However, when initial [3H]ouabain binding in tissues incubated in K+-free solutions was measured the stimulation of the initial rate of [3H]ouabain binding caused by insulin was markedly reduced. Incubating the apical side of the epithelium with either amiloride or Na+-free solutions also reduced or abolished the increase in the initial rate of [3H]ouabain binding caused by insulin. Equilibrium binding measurements showed that insulin did not increase the maximum number of [3H]ouabain-binding sites in tissues incubated with either normal K+ or K+-free solutions. These results indicate that the increase in the initial rate of [3H]ouabain binding under transporting conditions is due to an effect on the binding kinetics of ouabain, probably related to an increased rate of Na+ entry, rather than to an increase in the number of Na+-K+-ATPases in the basolateral membrane. Cycloheximide inhibited both the increase in Isc and the increase in the initial rate of [3H]ouabain binding caused by insulin in epithelia incubated in K+-containing solutions. However, cycloheximide was without effect on the initial rate of [3H]ouabain binding in insulin-treated tissues incubated in K+-free solution. This finding suggests that the cycloheximide-sensitive step of the action of insulin is related to Na+ delivery to the pump.

Effect of insulin on area and Na+ channel density of apical membrane of cultured toad kidney cells.

1. The stimulation of transepithelial Na+ transport caused by insulin in A6 cultured toad kidney cells was investigated by determination of membrane capacitance (Cm), short circuit current (Isc) and current fluctuation analysis. Values of Cm are proportional to membrane area while blocker-induced current fluctuation analysis provides an estimate of the number of active amiloride-sensitive Na+ channels in the apical membrane. 2. Insulin simultaneously increased Cm, Isc and Gt (transepithelial conductance) in epithelia incubated with Na(+)-containing solutions on both sides. 3. Analysis of 6-chloro-3,5-diaminopyrazine-2-carboxamide (CDPC)-induced noise showed that insulin increased the number of active Na+ channels in the apical membrane, without altering the single channel current. 4. When nystatin was used to permeabilize the apical membrane the impedance data revealed the presence of a second time constant. Analysis of these data indicated that the basolateral membrane capacitance (Cb) is much larger than the apical membrane capacitance (Ca). Insulin administered to nystatin-treated epithelia increased the values for both capacitances. 5. We suggest that the stimulation of transepithelial Na+ transport caused by insulin may be associated with the exocytotic delivery of transporters to the apical membrane.

Cell swelling activates a poorly selective monovalent cation channel in the apical membrane of toad urinary bladder.

We measured the effects of conditions that increase cell volume on ion movements through the Ca(2+)-blockable poorly selective monovalent cation channel of the apical membrane of the toad urinary bladder. Three conditions were studied: dilution of the basolateral solution, basolateral perfusion with solutions prepared with solutes of low reflection coefficient and dilution of the apical solution in bladders treated with oxytocin. All three procedures markedly increased K+ movements and elevated the plateau of the Lorentzian component of the power spectrum by enhancing ion currents through the Ca(2+)-blockable pathway. Simultaneously there was a large increase in Ca(2+)-sensitive conductance. The magnitude of this increased conductance strongly suggests that the stimulation of ion flow is due to increased ion permeability and not solely to increases in driving force across the apical membrane. We were not able to detect an increase in the movements of alkali-earth ions induced by the conditions that increase cell volume. We speculate that activation of the Ca(2+)-blockable channel may play an important role in the regulation of cell volume and/or in K+ homeostasis.

Cytochrome P-450 arachidonate metabolites affect ion fluxes in rabbit medullary thick ascending limb.

The medullary thick ascending limb of Henle's loop (mTALH) of the rabbit metabolizes arachidonic acid (AA) via a cytochrome P-450 (P-450) monooxygenase pathway to several products, of which the principal are 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) and 1,20-eicosatetraenedioic acid (20-COOH-AA). To understand their mechanism of action on alkali cation metabolism in mTALH cells, we have compared their effects with those of ouabain and furosemide. Incubation of rabbit isolated mTALH cells with either 1 mM ouabain or furosemide decreased K+ content from a control of 1,015 +/- 51 peq/micrograms protein to 717 +/- 41 and to 548 +/- 48 peq/micrograms protein, respectively, whereas they had opposite effects on Na+ content; from a control of 138 +/- 22 peq/micrograms protein, ouabain increased Na+ content to 357 +/- 37 peq/micrograms protein, and furosemide decreased it to 64 +/- 23 peq/micrograms protein. Preincubation with either 20-HETE (1 microM) or 20-COOH-AA (1 microM) decreased Na+ and K+, resembling furosemide in their effects on Na+ and K+ content. In other experiments we used monensin-treated cells to determine 86Rb uptake under conditions in which Na+ entry into the cell was not rate limiting. Under these conditions ouabain still inhibited 86Rb uptake, and the effect of AA was blocked. A major action of AA metabolites on Na(+)-K(+)-adenosinetriphosphatase was thereby excluded. Furthermore, AA metabolites did not inhibit Ba(2+)-sensitive 86Rb efflux, indicating that they do not act through K+ channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytochrome P450-dependent arachidonate metabolites affect renal transport in the rabbit.

Arachidonic acid (AA) inhibited 86Rb uptake in a dose-dependent manner in rabbit medullary tubules representing the thick ascending limb of the loop of Henle (mTALH). We have shown that CoCl2 treatment of rabbits reduced renal AA metabolism by cytochrome P450-dependent enzymes (P450). After CoCl2 treatment, AA was without effect on 86Rb uptake in mTALH cells. However, the two principal P450-AA metabolites produced by the mTALH, 20-HETE and 20-COOH-AA, inhibited 86Rb uptake in a dose-dependent manner in mTALH cells whether obtained from control or CoCl2-treated rabbits. These data support our hypothesis that CoCl2 treatment impairs ion transport function of mTALH cells by blocking cytochrome P450-AA metabolism, a blockade circumvented by the relevant P450-AA metabolites.

Urinary Ca2+ and the regulation of K+ secretion in toad bladder by neurohypophyseal hormones.

We measured net K+ fluxes in the isolated toad urinary bladder to determine whether neurohypophyseal hormones control K+ secretion in this tissue. To determine if the pathway involved in K+ secretion is similar to a Ca(2+)-blockable alkali cation channel in the apical membrane of toad bladder, previously described in electrophysiological studies, we measured K+ fluxes in either the presence or absence of Ca2+ in the mucosal bathing solution. Oxytocin enhanced K+ secretion in both cases; however, the enhancement was markedly greater in the absence of mucosal Ca2+. In other experiments the transepithelial voltage was held constant at a value of 120 mV (mucosa negative) to find whether hyperpolarization would enhance K+ secretion to the levels seen in Ca(2+)-free solutions. The response to oxytocin was markedly greater in the absence of mucosal Ca2+ even when the transepithelial voltage was continuously hyperpolarized. These observations suggest that the properties of the activated pathway are akin to those of the previously described Ca(2+)-blockable alkali cation channel. We also found that toad bladder urine often contained extremely low levels of Ca2+; therefore neurohypophyseal hormonal control of K+ transport across the bladder may play an important role in amphibian K+ balance.

Cyclic AMP increases electrical capacitance of apical membrane of toad urinary bladder.

We have measured the effects of oxytocin and three other compounds (chlorophenyl-thio-cyclic AMP, forskolin and theophylline) that increase cytoplasmic cyclic AMP on the impedance of the toad urinary bladder. Membrane capacitance was calculated from transepithelial impedance measured by a computerized sine wave method. All four agents increased tissue capacitance. Since in these tissues this parameter is proportional to apical membrane area our results suggest that cAMP can be a second messenger involved in the action of agents that promote fusion of exocytotic vesicles with the apical membrane.

Effect of cytochrome P450 arachidonate metabolites on ion transport in rabbit kidney loop of Henle.

In the medullary segment of the thick ascending limb of the loop of Henle (mTALH), arachidonic acid (AA) is metabolized by a cytochrome P450-dependent monooxygenase to products that affect ion transport. The linkage between changes in ion transport and AA metabolism in isolated cells of the mTALH was examined. AA produced a concentration-dependent inhibition of 86Rb uptake--an effect that was prevented by selective blockade of cytochrome P450 monooxygenases. Inhibition by cytochrome P450 blockade of the effect of AA on 86Rb uptake could be circumvented by addition of the principal products of AA metabolism in the mTALH.

Ca2+ entry through the apical membrane reduces antidiuretic hormone-induced hydroosmotic response in toad urinary bladder.

The role of Ca2+ in the regulation of antidiuretic hormone(ADH)-induced water permeability of the apical membrane of the toad urinary bladder was examined. The effects of modifying Ca2+ entry through the apical membrane of toad urinary bladders on the hydroosmotic water flow (phi H2O) and short circuit current (Isc) were measured. In most experiments the bladders were treated with small amounts of Ag+ (10(-7) mol/l) on the apical side. This treatment was used because previous experiments indicate that it markedly increases alkali-earth cation fluxes through an amiloride-insensitive cation channel in the apical membrane of the urinary bladder. Moreover, when Ca2+ is the major cation in the apical solution of these Ag(+)-treated bladders, Isc is mostly due to Ca2+ entry through the apical membrane. Ag+ increased Isc and simultaneously inhibited phi H2O in bladders perfused with Ca2+ solutions on the apical side. Addition of La3+ to the apical solution reversed the stimulation of Isc and the inhibition of phi H2O produced by Ag+. When bladders were perfused with Ca2(+)-free solutions on the apical side, addition of Ag+ did not inhibit phi H2O while the stimulation of cation movements through the amiloride-insensitive cation channel persisted. In bladders perfused with apical Ca2+ solutions and treated with chlorophenyl thio-cyclic adenosine monophosphate (ClPheS-cAMP) the addition of Ag+ did not inhibit phi H2O while it still increased Isc. Finally, addition of Ca2+ to the apical solution of bladders not treated with Ag+ reduced phi H2O.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of an apical cation-selective channel in function of tight epithelia.

Some features of oxytocin stimulation of a cation-selective channel of the apical membrane of amphibian tight epithelia were examined in an attempt to understand the channel's role in the regulation of epithelial transport. We first examined the ability of the channel to pass alkaline-earth cations. We found that oxytocin can stimulate the movement of alkaline-earth ions through the channel. This stimulation became greatly enhanced by treatment with Ag+. The stimulation of alkaline-earth movements is discussed together with recently reported experiments which suggest that the channel may be involved in K+ secretion. In addition we carried out comparative studies of the effects of oxytocin on the channel in a variety of epithelia obtained from different amphibians to examine whether the stimulation of ionic currents through the channel and the enhancement of hydrosmotic permeability caused by the hormone are linked. The results of our experiments showed that oxytocin activates the channel in the urinary bladders of Bufo marinus, and Rana catesbeiana as well as in the skin of B. marinus. It is well known that in all these tissues the hormone increases water permeability of the apical membrane. On the other hand, in skins of Rana catesbeiana, Rana pipiens, and Rana temporaria, where oxytocin does not have a hydrosmotic effect, the hormone did not increase the currents through the cation-selective channel.

Exocytotic events unrelated to regulation of water permeability in amphibian tight epithelia: effects of oxytocin, PMA and insulin on membrane capacitance, water and Na+ transport.

We measured the effects of oxytocin on capacitance and hydroosmotic water flow in the urinary bladder of the toad Bufo marinus and the skins of Rana pipiens and Rana temporaria. Oxytocin increased capacitance in all these tissues but stimulated hydroosmotic water flow only in the urinary bladder. We also measured the effects of oxytocin and PMA on the capacitance and hydroosmotic water flow of the toad urinary bladder. Both agents produced increases in membrane capacitance that were additive, however, PMA produced a stimulation of water flow that was only a fraction of that caused by oxytocin. Comparison of the effects of PMA and insulin in toad urinary bladder showed that in contrast with PMA, insulin did not increase membrane capacitance in this tissue. Moreover, insulin stimulated Isc in the urinary bladder while PMA produced an inhibition of variable magnitude. These results suggest that: (1) oxytocin can promote the fusion with the apical membrane of cytoplasmic membranes with or without water channels; (2) oxytocin and PMA stimulate the fusion with the apical membrane of cytoplasmic membranes originating in different pools; membranes in each pool have different water permeabilities and their insertion is controlled by different signals; (3) PMA and insulin act through different mechanisms in the toad urinary bladder.