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Dopamine D3R are widely expressed in basal ganglia where interact with D1R. D3R potentiate cAMP accumulation and GABA release stimulated by D1R in striatonigral neurons through "atypical" signaling. During dopaminergic denervation, D3R signaling changes to a "typical" in which antagonizes the effects of D1R, the mechanisms of this switching are unknown. D3nf splice variant regulates membrane anchorage and function of D3R and decreases in denervation; thus, it is possible that D3R signaling switching correlates with changes in D3nf expression and increases of membranal D3R that mask D3R atypical effects. We performed experiments in unilaterally 6‐hydroxydopamine lesioned rats and found a decrease in mRNA and protein of D3nf, but not of D3R in the denervated striatum. Proximity ligation assay showed that D3R‐D3nf interaction decreased after denervation, whereas binding revealed an increased Bmax in D3R. The new D3R antagonized cAMP accumulation and GABA release stimulated by D1R; however, in the presence of N‐Ethylmaleimide (NEM), to block Gi protein signaling, activation of D3R produced its atypical signaling stimulating D1R effects. Finally, we investigated if the typical and atypical effects of D3R modulating GABA release are capable of influencing motor behavior. Injections of D3R agonist into denervated nigra decreased D1R agonist‐induced turning behavior but potentiated it in the presence of NEM. Our data indicate the coexistence of D3R typical and atypical signaling in striatonigral neurons during denervation that correlated with changes in the ratio of expression of D3nf and D3R isoforms. The coexistence of both atypical and typical signaling during denervation influences motor behavior. [ABSTRACT FROM AUTHOR]

Abstract: Recent studies suggested the expression of CB2 receptors in neurons of the CNS, however, most of these studies have only explored one aspect of the receptors, i.e., expression of protein, messenger RNA, or functional response, and more complete studies appear to be needed to establish adequately their role in the neuronal function. Electron microscopy studies showed the presence of CB2r in asymmetric terminals of the substantia nigra pars reticulata (SNr), and its mRNA appeared is expressed in the subthalamic nucleus. Here, we explore the expression, source, and functional effects of such receptors by different experimental approaches. Through PCR and immunochemistry, we showed mRNA and protein for CB2rs in slices and primary neuronal cultures from subthalamus. GW833972A, GW405833, and JHW 133, three CB2r agonists dose‐dependent inhibited K+‐induced [3H]‐Glutamate release in slices of SNr, and the two antagonist/inverse agonists, JTE‐907 and AM630, but not AM281, a CB1r antagonist, prevented GW833972A effect. Subthalamus lesions with kainic acid prevented GW833972A inhibition on release and decreased CB2r protein in nigral synaptosomes, thus nigral CB2rs originate in subthalamus. Inhibition of [3H]‐Glutamate release was PTX‐ and gallein‐sensitive, suggesting a Giβγ‐mediated effect. P/Q Ca2+‐type channel blocker, ω‐Agatoxin‐TK, also inhibited the [3H]‐Glutamate release, this effect was occluded with GW833972A inhibition, indicating that the βγ subunit effect is exerted on Ca2+channel activity. Finally, microinjections of GW833972A in SNr induced contralateral turning. Our data showed that presynaptic CB2rs inhibit [3H]‐Glutamate release in subthalamo‐nigral terminals by P/Q‐channels modulation through the Giβγ subunit and suggested their participation in motor behavior. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The passive Na transport across the apical membrane of frog skin ( Rana catesbeiana) was studied under the following circumstances: (1) control conditions (sulfate Ringer's, K depolarised serosal membranes); (2) after blocking the active transport step with ouabain; (3) with an outward oriented Na current. The amiloride-induced Na current fluctuations were analysed to calculated the density of amiloride blockable channels and the current through one single channel. Despite the large reduction of the macroscopic current by oubain, the single channel current remained unchanged, while the number of amiloride blockable Na channels was reduced by a factor of eight. It is concluded from these observations that the earlier described reduction of the permeability of the apical membrane is caused by a decrease of the number of electrically conductive Na channels. The outward oriented single channel currents were less than 50% of the currents in the opposite direction. After ouabain, the number of Na channels was independent from the current direction. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

We measured the effects of conditions that increase cell volume on ion movements through the Ca-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-blockable pathway. Simultaneously there was a large increase in Ca-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 alkaliearth ions induced by the conditions that increase cell volume. We speculate that activation of the Ca-blockable channel may play an important role in the regulation of cell volume and/or in K homeostasis. [ABSTRACT FROM AUTHOR]

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-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 Ca in the mucosal bathing solution. Oxytocin enhanced K secretion in both cases; however, the enhancement was markedly greater in the absence of mucosal Ca. 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-free solutions. The response to oxytocin was markedly greater in the absence of mucosal Ca 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-blockable alkali cation channel. We also found that toad bladder urine often contained extremely low levels of Ca; therefore neurohypophyseal hormonal control of K transport across the bladder may play an important role in amphibian K balance. [ABSTRACT FROM AUTHOR]

The role of Ca 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 Ca entry through the apical membrane of toad urinary bladders on the hydroosmotic water flow Φ) and short circuit current ( I) were measured. In most experiments the bladders were treated with small amounts of Ag (10 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 Ca is the major cation in the apical solution of these Ag-treated bladders, I is mostly due to Ca entry through the apical membrane. Ag increased I and simultaneously inhibited Φ in bladders perfused with Ca solutions on the apical side. Addition of La to the apical solution reversed the stimulation of I and the inhibition of Φ produced by Ag. When bladders were perfused with Ca-free solutions on the apical side, addition of Ag did not inhibit Φ while the stimulation of cation movements through the amiloride-insensitive cation channel persisted. In bladders perfused with apical Ca solutions and treated with chlorophenyl thio-cyclic adenosine monophosphate (ClPheS-cAMP) the addition of Ag did not inhibit Φ while it still increased I. Finally, addition of Ca to the apical solution of bladders not treated with Ag reduced Φ. These results taken together with other findings in the literature suggest: (1) Ca entry through the Ag-treated amiloride-insensitive cation channel of the apical membrane inhibits Φ; (2) the effects of Ca entry are at a regulatory site that precedes the interaction of cAMP with the water channels; (3) it is also possible that Ca entry through the unmodified amiloride-insensitive cation channel may have some inhibitory effect on Φ. [ABSTRACT FROM AUTHOR]

We measured the effects of oxytocin (0.1 U/ml) on the current ( I) recorded through skins of Rana temporaria incubated with an isotonic K solution on the api al side while the transepithelial potential was clamped to zero. Under these conditions, I is carried by inward K movements. Oxytocin markedly stimulated this inward K current. When the spontaneous fluctuations were analyzed we found that oxytocin increased the plateau ( S) of the spontaneous Lorentzian component without modifying the corner frequency ( f). Addition of Ba to the mucosal solution blocked I both in the presence and absence of oxytocin. Moreover, with mucosal Ba a characteristic blocker-induced Lorentzian component appeared in the power spectrum. Analysis of this blocker-induced noise showed that oxytocin increased the number of active K channels in the apical membrane, while the changes in single channel current were in agreement with expected alterations of the electrochemical driving force. [ABSTRACT FROM AUTHOR]

Increasing [K] from 2.5 mmol/l to 115 mmol/l on the serosal side of the frog skin produces a rapid decrease of short-circuit current (I) that is followed, within a few minutes, by a recovery of I to near or above its control value. After isolation of the epithelium by a procedure involving collagenase treatment and physical removal of the corium, increasing serosal [K] still produced a depression of I but no significant recovery phase. By itself, collagenase treatment reduced but did not eliminate the recovery phase. The recovery phase was also markedly depressed by the beta-adrenergic blocker oxprenolol, but not by propranolol, atropine or indomethacin. Amiloride, given during the recovery phase, caused I to reverse to a small outward value. These results suggest that the recovery phase of I seen in the response to increased serosal [K] represents an increase in Na influx through amiloride-sensitive channels which is triggered by the release of an intermediary agent, possibly a beta-adrenergic agonist, from some structure in the corium. [ABSTRACT FROM AUTHOR]

We have measured the effects of concentrations of vanadate ranging between 0.01 and 10 mM on theNa efflux of frog sartorius muscles. The addition of vanadate had no effects when concentrations lower than 0.5 mM were used; higher concentrations increased Na efflux. The increase was abolished by the addition of ouabain (10M). In muscles pretreated with ouabain vanadate did not modify Na efflux. The stimulatory effects of vanadate on Na efflux were also observed in Na-free solutions indicating that the effux of vanadate was not caused mainly either by an increase in the exchange of Na for Na or by an increase in Na entry into the muscle. We also examined the effects of vanadate on muscles immersed in solutions containing 20 mM K; both vanadate and increased K produced stimulations of Na efflux that were additive. Similarly when the effects of vanadate and insulin were measured on the Na efflux of the same muscle, additive effects were found. As the ouabain-sensitive Na efflux in frog muscle is generally agreed to be due to the activity of the Na-K ATPase, our findings suggest that the net effect of vanadate in intact muscle cells is an increase in the activity of the Na pump. Since vanadate affects many enzymes it is quite possible that the stimulatory action is not due to a direct effect on the Na-K ATPase but may be mediated through an intermediary step. Regardless of the specific mechanism, it is evident that, our results as well as other findings in the literature, strongly indicate that Na pumping by intact cells can be increased by vanadate administration. Hence it is not justified to attribute the physiological modifications caused by vanadate administration to blockade of the Na-K ATPase unless the attribution is justified by specific experimental evidence. [ABSTRACT FROM AUTHOR]

We have compared some features of the resting and the insulin-stimulated uptake of α-aminoisobutyrate (AIB) in frog skeletal muscle. We found a substantial difference between the two processes, namely, that resting AIB uptake is Na-independent while the insulin-stimulated fraction of the AIB uptake is Na-dependent. Since the amino acid transport systems in frog skeletal muscle are poorly characterized, we have also surveyed some of their properties. One of the most interesting findings of this survey is that both the uptake and efflux of AIB are inhibited by low concentrations of PCMBS (parachloro-mercury-benzene sulfonic acid 5×10 m). In contrast, the carrier mediated transport of basic amino acids is neither inhibited by this mercurial agent nor accelerated by insulin. The action of PCMBS strongly suggests the presence of a critical sulfhydryl group in the amino acid carrier system utilized by AIB. This group is exposed to the outside solution since PCMBS penetrates cell membranes poorly, and in addition its inhibitory actions were reverted by agents that do not penetrate the cell membrane like albumin or glutathione. [ABSTRACT FROM AUTHOR]

The movements and distribution of lanthanum have been studied in frog skins treated on the outside with Ringer's solution to which 400MM urea had been added. Under these conditions,(140)La moved across the skin. This finding is in contrast with what is observed in control skins, which are impermeable to lanthanum. Examination with the electron-microscope of skins fixed during equilibration with outside hypertonic solutions plus lanthanum, showed penetration of the tracer from the outside into the intercellular spaces of all the inner layers of the epidermis. The occluding zonules (tight junctions) that normally prevent the inward movement of lanthanum beyond the outer border of the stratum granulosum (SG) are open. As a consequence, most intercellular spaces along the lateral cell surfaces of the SG were permeated by lanthanum throughout their extension. In skins treated with hypertonic solutions and then allowed to recover in normal Ringer's, the occluding zonules of the SG were impermeable to lanthanum, and(140)La did not move across the epidermis. These observations show that the increased skin permeability caused by outside hypertonic solutions results mainly from a reversible opening of tight junctions. [ABSTRACT FROM AUTHOR]

A simple method for the routine mounting of epithelia which greatly reduces or eliminates faulty sealing at the edge is described. The values of potential and resistance observed in the frog skin and toad urinary bladder are comparable to those found with more complicated methods designed to minimize edge damage. In agreement with observations in other epithelia, a direct relationship between short circuit and conductance is observed for frog skin and toad urinary bladder, implying that the relationship is a general feature of tight epithelia. Furthermore, the high values of transepithelial resistance imply that the lower limit for the paracellular shunt pathway resistance should be reset upwards. [ABSTRACT FROM AUTHOR]

In vivo activation of dopamine D3 receptors (D3Rs) depresses motor activity. D3Rs are widely expressed in subthalamic, striatal, and dendritic dopaminergic inputs into the substantia nigra pars reticulata (SNr). In vitro studies showed that nigral D3Rs modulate their neurotransmitter release; thus, it could be that these changes in neurotransmitter levels modify the discharge of nigro-thalamic neurons and, therefore, motor behavior. To determine how the in vitro responses correspond to the in vivo responses, we examined the effect of intra-nigral and systemic blockade of D3Rs in the interstitial content of glutamate, dopamine, and GABA within the SNr using microdialysis coupled to motor activity determinations in freely moving rats. Intranigral unilateral blockade of D3R with GR 103,691 increased glutamate, dopamine, and GABA. Increments correlated with increased ambulatory distance, non-ambulatory activity, and induced contralateral turning. Concomitant blockade of D3R with D1R by perfusion of SCH 23390 reduced the increase of glutamate; prevented the increment of GABA, but not of dopamine; and abolished behavioral effects. Glutamate stimulates dopamine release by NMDA receptors, while blockade with kynurenic acid prevented the increase in dopamine and, in turn, of GABA and glutamate. Finally, systemic administration of D3R selective antagonist U 99194A increased glutamate, dopamine, and GABA in SNr and stimulated motor activity. Blockade of intra-nigral D1R with SCH 23390 prior to systemic U 99194A diminished increases in neurotransmitter levels and locomotor activity. These data highlight the pivotal role of presynaptic nigral D3 and D1R in the control of motor activity and help to explain part of the effects of the in vivo administration of D3R agents. [ABSTRACT FROM AUTHOR]

Extensive damage to nigrostriatal dopaminergic neurons leads to Parkinson's disease (PD). To date, the most effective treatment has been administration of levodopa (L-DOPA) to increase dopaminergic tone. This treatment leads to responses that vary widely among patients, from predominantly beneficial effects to the induction of disabling, abnormal movements (L-DOPA induced dyskinesia (LID)). Similarly, experimental studies have shown animals with widely different degrees of LID severity. In this study, unilateral injections of 6-hydroxydopamine (6-OHDA) in the medial forebrain bundle (MFB) produced more than 90% depletion of dopamine in both the striatum and the substantia nigra reticulata (SNr) of rats. Population analysis showed that dopamine depletion levels were clustered in a single population. In contrast, analysis of abnormal involuntary movements (AIMs) induced by L-DOPA treatment of 6-OHDA-lesioned animals yielded two populations: one with mild LID, and the other with severe LID, which are also related to different therapeutic responses. We examined whether the severity of LID correlated with changes in dopamine 3 receptor (D3R) signaling because of the following: (a) D3R expression and the induction of LID are strongly correlated; and (b) dopaminergic denervation induces a qualitative change in D3R signaling in the SNr. We found that the effects of D3R activation on cAMP accumulation and depolarization-induced [3H]-gamma-aminobutyric acid ([3H]-GABA) release were switched. L-DOPA treatment normalized the denervation-induced changes in animals with mild LID. The D3R activation caused depression of both dopamine 1 receptor (D1R)-induced increases in cAMP production and depolarization-induced [3H]-GABA release, which were reversed to their pre-denervation state. In animals with severe LID, none of the denervation-induced changes were reversed. The finding that in the absence of identifiable differences in 6-OHDA and L-DOPA treatment, two populations of animals with different D3R signaling and LIDs severity implies that mechanisms intrinsic to the treated subject determine the segregation. [ABSTRACT FROM AUTHOR]