Phorbol esters and D2-dopamine receptors

4-beta-Phorbol-12,13-dibutyrate (PDBu), a powerful activator of protein kinase C (PKC), enhanced dopamine (DA) release evoked by electrical stimulation (1 Hz, 2 min) from the striatum and the prefrontal cortex of the rabbit. However, acetylcholine (ACh) release from the striatum (1 Hz, 2 min), was only enhanced slightly by PDBu. The increase in DA release induced by PDBu was reduced markedly at higher frequencies of stimulation. Sulpiride (10 microM) alone, a D2 DA-receptor antagonist, or combined with nomifensine (3 microM), a neuronal-uptake inhibitor, did not prevent PDBu-induced facilitation of DA release from prefrontal cortex or striatum. The D2 DA agonists (LY-171555, bromocriptine and apomorphine) inhibited in a concentration-dependent manner the stimulation-evoked overflow of DA and ACh from the striatum, and of DA from the prefrontal cortex. Pretreatment with PDBu antagonized the inhibitory effect of the three agonists on DA and ACh release. A reduction both in Emax and IC50 was observed in PDBu-treated slices. Removal of endogenous DA by pretreatment with reserpine and alpha-methyl-p-tyrosine, failed to prevent PDBu-induced antagonism of apomorphine effects on ACh release, indicating that the antagonism of agonist effects was not due to higher synaptic levels of endogenous DA. The inactive enantiomer of PDBu, 4-alpha-12,13-dibutyrate did not enhance DA release and failed to modify the effects of D2 agonists on DA release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sustained high release at rapid stimulation rates and reduced functional autoreceptors characterize prefrontal cortex dopamine terminals

The release of dopamine (DA) from mesocortical and nigrostriatal nerve terminal fields, as well as its modulation by auto- and heteroreceptors was investigated. Rabbit brain slices obtained from medial prefrontal cortex (PFC) and nucleus caudate (striatum) were prelabeled with [3H]DA in the presence of 0.3 microM desipramine. Neuronal depolarization was elicited by electrical stimulation. Higher stimulation-evoked overflow of [3H]DA (release) was observed from PFC than from striatal slices. At 0.3 Hz (120 pulses) release from the PFC was 60% higher than from the striatum, and at higher frequencies (10 Hz and 120 or 1200 pulses) the fraction of tissue radioactivity released from the PFC was 550% greater than that released from the striatum. These differences were not eliminated by blockade of autoreceptors with haloperidol, or by inhibition of neuronal uptake with nomifensine. These results suggest that the coupling between neuronal depolarization and DA release is more efficient in the PFC than in the striatum. This may allow the PFC terminals to sustain neurotransmission under continuous fast firing. Selective D2 agonists, as well as nonselective DA agonists, inhibited DA release in a concentration-dependent fashion from the PFC and the striatum. Their effects were blocked by l-sulpiride or haloperidol. SKF 38393, a selective D1 agonist, produced a small facilitation of release from both regions; its effects were blocked by SCH 23390 (a selective D1 antagonist). The latter was ineffective on its own. The maximal degree of inhibition of release produced by apomorphine, bromocriptine and LY-171555 was lower in the PFC than in the striatum; these differences were accentuated greatly at high stimulation rates. When the slices were stimulated at frequencies comparable to the "in vivo" firing rates for each neuronal group, apomorphine and LY-171555 were much weaker in inhibiting DA release from the PFC (10 Hz) than from the striatum (3 Hz). In the striatum, strong modulation of DA release by endogenous DA was observed; whereas little modulation was seen in the PFC. Nomifensine produced larger increases in the stimulation-evoked overflow of DA from PFC and there was no synergistic interaction between nomifensine and haloperidol in this structure. In the striatum, marked facilitation of DA overflow was observed when nomifensine and haloperidol were given together. Furthermore, haloperidol per se facilitated DA release from both brain regions; however, the degree of facilitation was frequency dependent in the striatum, but not in the PFC.(ABSTRACT TRUNCATED AT 400 WORDS)     

Frequency-dependent muscarinic receptor modulation of acetylcholine and dopamine release from rabbit striatum

The effects of muscarinic receptor activation on the electrically evoked release of [3H]dopamine (DA) and [14C]acetylcholine (ACh) or [3H]ACh were investigated in rabbit striatal slices. Release was measured in the presence of 10 microM hemicholinium and 1 microM sulpiride to block choline uptake and prevent the effects of released DA on DA receptors modulating release. Stimulation (120 pulses, 20 mA, 2 msec) at 0.3, 3 and 10 Hz produced (3H or 14C) ACh release that sharply declined with increasing stimulation frequency. A flat frequency-release curve was obtained for DA. Oxotremorine (OXO), a direct muscarinic agonist (1-100 microM), produced a concentration-dependent inhibition of ACh release, inversely related to stimulation frequency, at a fixed number of pulses (120). When the number of pulses was modified to produce similar amounts of ACh release (20 pulses at 0.1 Hz, 39 pulses at 0.3 Hz, 120 pulses at 3 Hz and 350 pulses at 10 Hz), much greater inhibition of ACh release by OXO (0.3 and 3 microM) was obtained with lower frequencies and lower number of pulses. Physostigmine, an acetylcholinesterase inhibitor, decreased ACh release with an inverse relationship to stimulation frequency. Atropine (1 microM), a selective muscarinic antagonist, enhanced the release of ACh more at 10 Hz than at 0.3 and 3 Hz and completely antagonized the effects of OXO (10 microM) and physostigmine (1 microM) at all three stimulation frequencies. OXO (3 and 10 microM) enhanced DA release at 3 Hz. Physostigmine (1 microM) and atropine (1 microM) had no effect on DA release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein kinase C translocation in rat stomach fundus: effects of serotonin, carbamylcholine and phorbol dibutyrate

In an effort to characterize serotonergic receptor activation in rat stomach fundus, the potential role of protein kinases, more specifically protein kinase C (PKC), in serotonin-induced contraction of rat stomach fundus was examined. Staurosporine, a potent, but nonselective, inhibitor of protein kinases, attenuated basal, membrane-bound PKC activity in rat stomach fundus (IC50 = 10 nM). Although staurosporine (3-100 nM) produced a concentration-dependent inhibition of contractions elicited by serotonin (which does not increase phosphatidylinositol hydrolysis in the fundus), carbamylcholine (an agent stimulating phosphatidylinositol hydrolysis), and phorbol 12,13-dibutyrate (PDBu; a phosphatidylinositol-independent activator of PKC translocation), it was a more potent inhibitor of contractions produced by serotonin and PDBu than by carbamylcholine. Potassium chloride-induced contractions were attenuated minimally by staurosporine. These results raised the possibility that serotonin might exert an effect on protein kinase activity by a phosphatidylinositol-independent mechanism. Focusing on PKC, serotonin's ability to translocate PKC from cytosol to membrane in rat fundus was examined. Concentrations of serotonin (0.1-10 microM) which maximally contracted rat fundus did not translocate PKC. However, PDBu (10 nM-1 microM) and carbamylcholine (0.1-10 microM) significantly increased membrane-bound PKC activity. These results: 1) demonstrate that translocation of PKC occurred in rat stomach fundus in response to some, but not all, contractile agonists; 2) are consistent with the possibility that contraction of rat stomach fundus by carbamylcholine and PDBu may be related to increased membrane-bound PKC activity; and 3) indicate that serotonin-induced contraction, although potently blocked by staurosporine, did not result from PKC translocation in the rat stomach fundus.     

Protein kinase C and vascular smooth muscle contractility: effects of inhibitors and down-regulation

We have compared two different methods of attenuating protein kinase C (PKC) activity in vascular smooth muscle. First, the effects of two purported PKC inhibitors, staurosporine (stauro) and H-7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride], were examined on contractility of isolated, intact canine femoral artery. In arterial rings stauro was equipotent in relaxing contractions induced by phenylephrine (PE), phorbol-12,13-dibutyrate (PDBu) and KCl (IC50, 0.31 +/- 0.19; 0.35 +/- 0.2; and 0.34 +/- 0.16 microM). H-7, in comparison, was markedly less potent than stauro (IC50, 0.67 +/- 0.2, 2.33 +/- 0.24; and 6.5 +/- 5.5 microM for PE, PDBu and KCl, respectively). Pretreatment of tissues with 1 microM stauro suppressed tension development almost completely when PE and PDBu were the contractile agonists, and partially in K(+)-depolarized rings. H-7, in contrast, had no inhibitory effect on agonist-induced contraction. Neither basal nor K(+)-stimulated calcium influx was affected by 10 microM stauro. Second, prolonged exposure of canine carotid arterial rings to PDBu (1-100 nM for 24 hr), a means of depleting PKC from the tissue, caused dose-dependent attenuation of agonist-induced contractions. Preincubation with 100 nM PDBu caused complete inhibition of tension induced by norepinephrine (NE) and serotonin and partial inhibition of PDBu- and KCl-induced contractions. Lowering the concentration of PDBu during preincubation to 30, 10 or 1 nM reduced markedly the inhibitory effects. The inactive phorbolester 4 alpha-phorbol-12,13-didecanoate (4 alpha-PDD) had no effect on agonist-induced contractions. PKC activity was determined in rings contracted isometrically with PDBu or NE after prolonged exposure to vehicle, 4 alpha-PDD or PDBu.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of protein kinase C inhibitors on the actions of phorbol esters on vascular tone and adrenergic transmission in the isolated rat kidney

The effect of protein kinase C (PKC) inhibitors 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine (H-7), polymyxin B (PMB), D-sphingosine (SPH), sangivamycin (SNG) and staurosporin (ST) on the action of PKC activators phorbol 12,13-dibutyrate (PDBu) and 12-o-tetradecanoylphorbol-13-acetate (TPA), on adrenergic neuroeffector events was investigated to determine the contribution of PKC in adrenergic transmission in the rat kidney. Infusion of TPA (5 x 10(-6) mM) or PDBu (6 x 10(-6) mM) produced renal vasoconstriction and enhanced the overflow of tritium elicited by periarterial renal nerve stimulation (RNS) (2 Hz) in the isolated rat kidney perfused with Tyrode's solution and prelabeled with [3H]norepinephrine. H-7 (2.7 x 10(-3) mM) and ST (2 x 10(-5) mM) did not alter RNS-induced overflow of tritium but attenuated the vasoconstrictor response to RNS and exogenous NE. PMB (1 x 10(-8) mM) and SPH (3.3 x 10(-4) mM) but not SNG (3.3 x 10(-3) mM) attenuated the RNS-induced overflow of tritium but increased the basal renal vascular tone and enhanced the vasoconstrictor response to RNS and exogenous NE. H-7, PMB, SPH, SNG or ST failed to alter the effects of PDBu to increase basal vascular tone and the overflow of tritium and the increase in renal vasoconstriction to RNS. PMB at 1 x 10(-9) mM but not at 1 x 10(-8) mM and SPH (3.3 x 10(-4) mM) but not H-7, SNG or ST inhibited the effect of TPA to increase the overflow of tritium. The effect of TPA on the vasoconstrictor response to RNS or to increase basal vascular tone was not altered by PKC inhibitors. These data suggest that in the rat kidney, PKC is either resistant to the actions of H-7, PMB, SPH, SNG and ST, or PDBu and TPA produce renal vasoconstriction and facilitate adrenergic transmission by a mechanism unrelated to PKC activation.     

Inhibition of cAMP accumulation by kappa-receptor activation in isolated iris-ciliary bodies: role of phosphodiesterase and protein kinase C

The present study was designed to examine the roles of protein kinase C (PKC) and phosphodiesterase (PDE) in modulating the action of kappa receptor stimulation on cAMP accumulation in isolated iris-ciliary bodies (ICBs) of New Zealand White rabbits. The kappa receptor agonist, (+/-)-1-(3,4-dichlorophenyl)acetyl-2-(1-pyrrolidinyl)methylpiperidine (BRL-52537) (BRL), and the PKC activator, phorbol 12,13-dibutyrate (PDBu), both caused a concentration-dependent inhibition of forskolin-stimulated cAMP production. The inhibitory effect of BRL on cAMP levels was significantly reduced in the presence of the selective kappa receptor antagonist, norbinaltorphimine (10(-6) M), but the effect of PDBu was not, thus supporting the involvement of kappa-opioid receptors in the response to BRL. In the presence of 3-isobutyl-1-methylxanthine or rolipram (10(-5) M), the inhibitory effect of BRL or PDBu (10(-6) M) on cyclic AMP accumulation was abolished. In the presence of the selective PKC antagonist, chelerythrine (10(-6) M), the inhibitory effect of PDBu or BRL (10(-6) M) was significantly reduced. Direct measurement of PDE activity demonstrated the ability of BRL and PDBu (10(-6) M) to augment the activity of these enzymes. Preincubation of ICBs with rolipram (10(-5) M) or chelerythrine (10(-6) M) caused significant reversal of both BRL- and PDBu-induced increases in PDE activity. These results indicate that stimulation of PKC and PDE4 activity is part of the complex mechanism whereby kappa-opioid receptor agonists reduce levels of cAMP in the rabbit ICB. This mechanism of action could contribute to the ability of kappa-opioid agonists to suppress aqueous flow rate and to lower intraocular pressure.     

Phorbol-12,13-dibutyrate-induced vasoconstriction in vivo: characterization of response in genetic hypertension

To assess the role of protein kinase C (PKC) in the control of vessel tone in vivo in genetic hypertension, the vascular effects of phorbol-12,13-dibutyrate (PDBu), a PKC activator, was measured in the autoperfused hindlimb of reserpinized spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). PDBu infusion (1-3000 ng/kg/min) into the hindlimb elevated perfusion pressure in a dose-related manner. Vasoconstriction response characteristics (latency, T1/2 to peak effect, decay of effect) of PDBu were significantly longer (2- to 10-fold) than that produced by membrane receptor agonists; phenylephrine, SKF 89748, a lipophilic alpha-1 agonist, angiotensin II and 5-hydroxytryptamine. The tonic vasoconstriction induced by PDBu was not antagonized by prazosin, rauwolscine, cyproheptadine, [Sar1lle8]-angiotensin II but was inhibited reversibly by microbial PKC-inhibitors, K252a and staurosporine at concentrations (1.56-2.8 micrograms/kg/min) which did not block vasoconstriction by phenylephrine or 5-hydroxytryptamine. The EC50 for PDBu was identical in SHR and WKY. However, the maximal response to PDBu was significantly greater in SHR compared to WKY. Staurosporine lowered mean arterial pressure equally in SHR (20%) and WKY (17%) but reduced perfusion pressure in SHR (13%) to a slightly greater extent than in WKY (5%). Unlike the in vivo response, aortic rings removed from SHR were more sensitive to cumulative doses of PDBu than rings from WKY. It is concluded that PDBu-vasoconstriction in vivo is mediated largely through activation of PKC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of protein kinase C sensitizes human VR1 to capsaicin and to moderate decreases in pH at physiological temperatures in Xenopus oocytes

The two-electrode voltage-clamp technique was used to evaluate the effect of protein kinase C (PKC) activation on ion current flow in Xenopus laevis oocytes injected with cRNA coding for the human vanilloid receptor (VR1). In the presence of 30 nM phorbol-12,13-dibutyrate (PDBu), current evoked by an effective concentration (EC(30)) of capsaicin (CAP) was potentiated by 638+/-117% (n=8). PDBu exhibited an EC(50) of about 17+/-3 nM for this effect (n=8). Potentiation was not observed when VR1 expressing oocytes were exposed to both 30 nM PDBu and 1 microM staurosporine. In the presence of 300 nM PDBu, the EC(50) for CAP shifted from 899+/-78 to 139+/-2 1 nM (n=11 and 5, respectively). In the presence of 30 nM PDBu, the maximal current amplitude evoked by application of CAP increased by 86+/-21% (n=10), in a staurosporine sensitive manner. Application of 1 microM PDBu alone elicited a capsazepine sensitive current within 3 min of exposure. This effect was observed in the absence of previous exposure of the oocyte to CAP and was abolished in the presence of 1 microM staurosporine. No current was elicited during a 10 min application of 300 nM PDBu, the longest interval assessed. Prior to 30 nM PDBu exposure, no current was evoked at temperature ramps from room temperature (22-23 degrees C) up to 37 degrees C at pH 6.8, 7.0, or 7.4. Following PDBu treatment, VR1 mediated current was evoked at 26 degrees C at pH 7.0. Likewise, following 30 nM PDBu treatment, current was evoked by application of pH 6.8 alone and a further increase in current amplitude was evoked by heat at 24 degrees C in a staurosporine sensitive manner. These data provide direct evidence that PKC activation can increase VR1 current evoked by candidate physiological activators, pH and heat. This observation provides an empirical foundation for explaining some types of inflammatory pain in terms of PKC activation, small decreases in tissue pH levels, and small increases in skin temperature, all of which can accompany inflammatory conditions.     

Comparative dopamine-cholinergic mechanisms in the olfactory tubercle and the striatum: effects of metoclopramide

The olfactory tubercle (OT) is a limbic structure containing high dopamine (DA) and acetylcholine (ACh) concentrations. We performed a comparative study of the DA-ACh interactions, the efficacy of autoreceptor control and the effects of metoclopramide in the OT and the nucleus caudate (striatum). Rabbit brain slices from both regions of the same animal were prelabeled with radioactive DA and/or choline and then superfused. Comparable magnitude of DA and ACh release was evoked by electrical stimulation from both regions. DA release was unaltered, whereas ACh release was inversely related to the stimulation frequency, both in OT and striatum. Apomorphine (APO), a D1-D2 agonist, an LY-171555 (LY), a D2 agonist, inhibited DA and ACh release from OT and striatum with similar EC50 and Emax (maximal percentage of inhibition). However, the maximal degree of inhibition of ACh release achieved with APO, LY or DA in the OT was only one-half that observed in the striatum. In both regions, the inhibitory effects of DA agonists on DA and ACh release were reduced markedly when the number of electrical pulses and/or the frequency of stimulation were increased. l-Sulpiride, a DA D2 antagonist, increased the evoked release of DA and ACh from OT in direct relationship with the frequency of stimulation. In the OT, increases in synaptic DA achieved by administration of amphetamine or by blockade of the neuronal uptake pump with nomifensine inhibited the evoked release of ACh. Again these drug treatments produced only a 40 to 50% inhibition of ACh release. SKF 38393, a D1 agonist, had no effect per se on DA or ACh release in OT slices from control or from reserpine-treated animals (2 mg/kg s.c. for 3 or 7 days). With the exception of one specific dose combination, coadministration of SKF 38393 and LY produced no additive or synergistic effects on DA or ACh release from OT. APO- and LY-induced inhibition of DA and ACh release were antagonized by l-sulpiride. However, 300 nM SCH 23390, a D1 antagonist, reduced APO inhibition of DA and ACh release without affecting the inhibitory action of LY on DA and ACh release. Metoclopramide, "a DA antagonist with poor limbic activity", had a similar affinity for OT (pA2: 7.59) and striatal (pA2: 7.59) DA autoreceptors. Its antidopaminergic efficacy on DA receptors modulating ACh release from OT and striatum was also compared.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential Effects of Protein Kinase C Activators and Inhibitors on alpha- and beta-Adrenoceptor-mediated Positive Inotropic Effect in Isolated Rabbit Papillary Muscle

BACKGROUND: A number of novel agents that activate or inhibit protein kinase C (PKC) in vitro have been developed to evaluate the physiologic role of PKC in regulation of cellular function. However, most of the PKC inhibitors also affect the protein kinase A, and the effects of these agents in intact myocardium remain still controversial. The present study was carried out to examine the effects of these agents on the positive inotropic effect (PIE) medicated by alpha- and beta-adrenoceptors in isolated rabbit papillary muscle. METHODS AND RESULTS: A potent PKC activator, phorbol 12, 13-dibutyrate (PDBu) at 10 and 30 nM, induced a significant PIE. PDBu at 3 nM and higher inhibited the alpha-mediated PIE and abolished it at 100 nM without affecting the beta-mediated PIE. Phorbol 12-myrisate 13-acetate (PMA) and 1-oleyl-2-acetyl-sn-glycerol (OAG) elicited a similar selective inhibitory action on the alpha-mediated PIE. The PIE of PDBu was abolished by chelerythrine and partially inhibited by staurosporine, but H-7 or calphostin-C did not affect the PIE. These PKC inhibitors consistently inhibited the alpha-mediated PIE by 20-30% at concentrations that they did not affect the beta-mediated PIE. None of the PKC inhibitors influence the PDBu-induced inhibitory action on the alpha-mediated PIE, an indication that they failed to reach the site of the inhibitory action of PDBu. CONCLUSION: Selective modulation by the PKC activators and inhibitors of the alpha-mediated PIE with little effect on the beta-mediated PIE implies that the activation of PKC has a physiological relevance to the alpha-mediated PIE. However, the externally administered PKC activators do not mimic the effect of diacylglycerol that is generated endogenously by alpha-stimulation. By contrast, externally applied PKC inhibitors selectively antagonize the alpha-adrenoreceptor-mediated PIE in rabbit ventricular myocardium.     

A detailed study on the effects of protein kinase C activation on alpha-2 adrenoceptor-coupled modulation of norepinephrine release in hippocampus.

The question was studied whether there is a direct link between protein kinase C and presynaptic alpha-2 adrenoceptors regulating depolarization-induced norepinephrine (NE) release. Effects of the protein kinase C activator 4 beta-phorbol 12,13-dibutyrate (4 beta-PDB) on electrically evoked [3H]NE release were investigated in rabbit and rat hippocampus. Release evoked with 4 pulses/100 Hz (POP stimulation; i.e. under conditions virtually free of autoinhibition), was increased by 4 beta-PDB in a comparable manner in both species. Conversely, the alpha-2 adrenoceptor agonist clonidine diminished POP-induced [3H]NE release in a concentration-dependent manner. The net effects of clonidine were of a similar magnitude up to near maximal concentrations, irrespective of whether or not the 4 beta-PDB was present. Correspondingly, the net effect of 4 beta-PDB remained unchanged under these conditions. An impairment of the net effect of 4 beta-PDB was only seen at higher concentrations of clonidine. Concurrent addition of the alpha-2 adrenoceptor antagonist yohimbine and 4 beta-PDB enhanced release elicited with 36 pulses/3 Hz (i.e., in presence of autoinhibition), in a manner which was at least additive. Taken together, the above data exclude a direct link between presynaptic alpha-2 adrenoceptors and protein kinase C and restrict a functional interaction to very distinctive conditions.     

Regulation of alpha1-adrenoceptor-mediated contractions of the uterine artery by protein kinase C: role of the thick- and thin-filament regulatory pathways

Previously we demonstrated that activation of protein kinase C (PKC) enhanced alpha(1)-adrenoceptor-induced contractions in nonpregnant uterine arteries (NPUA) by increasing the Ca(2+) sensitivity but that it inhibited the contractions in pregnant uterine arteries (PUA) by decreasing intracellular Ca(2+) mobilization. The present study tested the hypothesis that PKC activation differentially regulated the thick- and thin-filament regulatory pathways in alpha(1)-adrenoceptor-induced contractions of NPUA and PUA in sheep. Simultaneous measurements of contractions and phosphorylation levels of 20-kDa regulatory myosin light chain (LC(20)) in the same tissue revealed that the PKC activator phorbol-12,13-dibutyrate (PDBu) inhibited phenylephrine-induced phosphorylation of LC(20) and contractions in PUA. In NPUA, PDBu significantly potentiated phenylephrine-induced contractions without significantly changing phosphorylation levels of LC(20). Further studies in NPUA demonstrated that PDBu-mediated potentiation of phenylephrine-induced contractions was associated with a significant increase in phosphorylation levels of extracellular signal-regulated kinase (ERK(42/44)) and caldesmon-Ser(789), measured simultaneously with the tension in the same tissue. In addition, the ERK(42/44) inhibitor PD98059 [2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one] and the actin polymerization inhibitor cytochalasin B produced a concentration-dependent inhibition of PDBu-mediated potentiation of phenylephrine-induced contractions in NPUA. The results suggest that activation of PKC inhibits alpha(1)-adrenoceptor-mediated contractions in PUA through down-regulation of the thick-filament pathway and decreased myosin light chain phosphorylation, but that it enhances the contractions in NPUA through its effect on the thin-filament regulatory pathway and activation of ERK/caldesmon and actin polymerization.     

Changes in sensitivity of release modulating dopamine autoreceptors after chronic treatment with haloperidol

The release of recently taken up [3H]dopamine ([3H]DA) elicited by electrical stimulation (3 Hz, 2 min, 16 mA) from slices of the rabbit caudate nucleus is inhibited by apomorphine (0.01-0.1 microM) in a concentration-dependent manner. This action is mediated through the activation of presynaptic inhibitory DA autoreceptors. The inhibition of [3H]DA release by apomorphine (0.1 microM) was antagonized 2 hr, but not 24 hr after the single administration of haloperidol (1 mg/kg s.c.). After 2 days of withdrawal after 28 days of chronic treatment with haloperidol (1 mg/kg s.c.) once daily, apomorphine (0.01-0.1 microM) was more effective in inhibiting [3H]DA release elicited by electrical stimulation when compared with rabbits injected chronically with either the vehicle for haloperidol or with saline. In superfused slices of the rabbit caudate nucleus, exposure to S-sulpiride (0.1 and 1 microM) increased in a concentration-dependent manner the release of [3H] DA elicited by electrical stimulation. After 28 days of chronic treatment with haloperidol, the facilitation of [3H]DA release by S-sulpiride was significantly reduced when compared with the controls. The inhibition of central noradrenergic transmission by DA receptor agonists was studied in hypothalamic slices prelabeled with [3H]norepinephrine ([3H-NE]). Apomorphine (0.01-1 microM) inhibited the electrically evoked (5 Hz, 2 min, 26 mA) release of [3H]NE from hypothalamic slices of untreated rabbits. The sensitivity to the inhibitory effect of apomorphine on [3H]NE overflow remained unaffected after 2 days of withdrawal following 28 days of chronic treatment with haloperidol. In summary, our results indicate that chronic haloperidol administration induces changes in sensitivity of the DA autoreceptors regulating dopaminergic neurotransmission but does not affect the sensitivity of DA receptors modulating NE release in the central nervous system. These results suggest that the DA autoreceptors that regulate dopaminergic neurotransmission may play a physiological role in the modulation of transmitter release and consequently are susceptible to the development of changes in sensitivity after chronic receptor blockade. The possible implication of changes in sensitivity of the DA autoreceptor during the treatment of schizophrenia with neuroleptics is discussed.     

Role of protein kinase C in electrical-stimulation-induced neuronal nitric oxide release in mesenteric arteries from hypertensive rats

This study examines the influence of hypertension on neuronal nitric oxide (NO) release and its modulation by protein kinase C (PKC). For this purpose, mesenteric segments without endothelium were obtained from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs), and neurogenic NO release induced by electrical field stimulation (EFS) was examined in these segments. EFS induced frequency-dependent contractions. The NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) and the sensorial neurotoxin capsaicin increased EFS-induced contractions in SHR segments, but did not affect these contractions in segments from WKY rats. In segments from SHRs, the increase in EFS-induced response by capsaicin was further increased by the combination of capsaicin and L-NAME. EFS-induced contractions in SHR arteries were unaltered by the protein synthesis inhibitor cycloheximide or by 2-amine-5,6-dihydro-6-methyl-4H-1,3-tiazine (AMT), an inhibitor of inducible NO synthase, and increased by the guanylate cyclase inhibitor Methylene Blue. In these arteries, capsaicin plus the PKC inhibitor calphostin C increased the contractions elicited by EFS; the addition of L-NAME did not affect this increase. Phorbol 12,13-dibutyrate (PDBu) did not modify the response to EFS in these arteries pretreated with capsaicin, although a combination of PDBu and L-NAME was effective. These results indicate that, in mesenteric arteries, EFS induces the release of NO from perivascular nitrergic nerves and of neuropeptides from sensory nerves, but only in hypertensive rats. The NO released is synthesized by constitutive neuronal NO synthase in a manner that is positively modulated by PKC, an enzyme that seems to be activated in hypertension.     

Dopamine efflux from striatal slices after intracerebral 6-hydroxydopamine: evidence for compensatory hyperactivity of residual terminals

In this study, we have examined the spontaneous efflux and stimulation-induced overflow of endogenous dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) from striatal slices prepared from adult rats. Partial destruction of striatal DA terminals with 6-hydroxydopamine reduced the electrically evoked overflow of DA and DOPAC, but by less than the DA content of the tissue slices. This resulted in an increase in the fractional overflow of DA and DOPAC, a measure of overflow from residual dopaminergic terminals. The increase in fractional DA overflow was related to the lesion size, reaching 7-fold with DA depletions of greater than 90%. Inhibition of DA re-uptake with nomifensine (10 microM) increased DA overflow and reduced DOPAC overflow by an equivalent amount, indicating that a portion of the DA released by electrical stimulation is recaptured and metabolized before appearing as DOPAC in the superfusate. However, 6-hydroxydopamine lesions further elevated fractional DA overflow above control even in the presence of nomifensine, suggesting that slices prepared from lesioned animals exhibit an increase in DA release from residual dopaminergic terminals. DA overflow was enhanced by the DA receptor antagonist sulpiride (1 microM) in control tissue but not in slices prepared from lesioned animals, suggesting that increased efflux per terminal was accompanied by reduced autoinhibition of release. Over a range of firing frequencies typical of nigrostriatal neurons in vivo (2-8 Hz), fractional DA overflow per pulse from lesioned slices was 3-fold higher than control overflow; however, fractional overflow per pulse was reduced from lesioned but not control slices when slices were exposed to a higher frequency (12 Hz). Thus, the lesion appeared to have increased DA release at moderate frequencies, but had reduced the effective range of frequencies over which the DA terminals could operate. Finally, 3-iodotyrosine (2 mM), an inhibitor of tyrosine hydroxylase, reduced DA overflow from intact slices, but completely abolished overflow from lesioned slices, suggesting that 6-hydroxydopamine had increased the dependence of DA efflux on a sustained rate of DA synthesis. Taken together, these data suggest that after lesioning with 6-hydroxydopamine, DA released per pulse from residual terminals is increased relative to control, so long as the stimulation frequency is within the physiological range. This increase in release may serve a compensatory function, maintaining dopaminergic control over striatal function despite extensive loss of DA neurons.     

Effects of d-amphetamine and dopamine synthesis inhibitors on dopamine and acetylcholine neurotransmission in the striatum. II. Release in the presence of vesicular transmitter stores

The release of endogenous dopamine (DA) elicited by electrical stimulation and by d-amphetamine (AMPH) from superfused striatal slices of untreated rabbits was examined. AMPH (0.3-10 microM) produced a concentration-dependent increase in basal DA efflux (30-fold increase at 10 microM) and stimulation-evoked (SE) DA overflow (11-fold increase at 10 microM). Although AMPH had little effect on the basal efflux of dihydroxyphenylacetic acid (DOPAC), the drug was an effective inhibitor of the SE overflow of the DA metabolite (66% inhibition at 0.3 microM). AMPH increased significantly the total basal efflux of endogenous compounds (DA + DOPAC) only at high concentrations (3-10 microM) whereas the total SE overflow of total endogenous compounds was decreased at all concentrations of AMPH tested. AMPH inhibited SE [3H]acetylcholine (ACh) release in a concentration-dependent manner (71% inhibition at 10 microM). Inhibition of DA synthesis with alpha-methyl-p-tyrosine (100 microM) or 3-iodotyrosine (100 microM) reduced both the basal efflux and SE overflow of endogenous DA and DOPAC; synthesis inhibition had greater effects on the SE overflow. Neither synthesis inhibitor altered SE [3H]ACh release. alpha-Methyl-p-tyrosine and 3-iodotyrosine reduced the absolute values of the basal efflux and SE overflow of DA elicited by AMPH by approximately 60%; however, the inhibition of SE [3H]ACh release produced by AMPH was attenuated only slightly (approximately 20%). Synthesis inhibitors also reduced tissue DA levels (approximately 30%). These results suggest that: basal efflux of endogenous DA from superfused rabbit striatal slices may derived both from DA newly synthesized in the cytoplasm and from spontaneous leakage of DA from storage vesicles. In addition, synthesis may provide a continuous supply of DA to vesicles that are used for exocytotic DA release during electrical stimulation. However, the depletion of tissue DA produced by synthesis inhibitors as well as other extraneous pharmacological actions of these drugs makes firm conclusions difficult. AMPH increases the synaptic concentration of DA by accelerating the basal efflux as well as the SE overflow of unchanged DA. At concentrations less than 1 microM AMPH has no effect on basal efflux of DA or DOPAC but reduces SE overflow of DOPAC via an unknown mechanism. At higher concentrations (greater than or equal to 1 microM) acceleration of carrier-mediated DA efflux coupled with displacement of DA from vesicular stores, as well as interference with the uptake of exocytotically released DA produces a marked increase in synaptic DA which in turn inhibits SE ACh release.(ABSTRACT TRUNCATED AT 400 WORDS)     

Neurotensin-induced dopamine release in vivo and in vitro from substantia nigra and nucleus caudate

We compared the dopamine (DA) releasing effects of neurotensin (NT) from cell bodies (substantia nigra) and nerve terminals (nucleus caudate). In rats implanted with push-pull cannula, NT induced DA release from substantia nigra and nucleus caudate. NT was more potent in releasing DA from the substantia nigra than from the nucleus caudate (EC50%, 1.1 microM in substantia nigra and 9.8 microM in nucleus caudate). In vitro, in superfused rabbit brain slices, NT enhanced the depolarization-evoked release of DA and exerted a direct releasing effect. The latter was greater in the substantia nigra, and the former in the nucleus caudate. The direct releasing effect of NT was not inhibited, but enhanced by nomifensine (3 microM). Sulpiride, a D2 DA receptor antagonist, failed to modify NT-induced DA release; in addition, NT did not affect the inhibition of DA and acetylcholine release produced by LY-171555, a D2 DA agonist. In both the substantia nigra and the nucleus caudate, desensitization to the releasing effect of NT was observed, either after 2.5, 5, or 10 min of exposure to the peptide. A synergistic interaction on DA release was observed between NT and potassium (K+), and between NT and electrical stimulation. Greater synergism was observed with high extracellular K+. Pretreatment of striatal slices with 15 mM K+ produced a 9-fold enhancement of NT-induced DA release. When K+ (25 mM, 2 min) was given together with NT there was a 2- to 3-fold increase in DA release compared to the release evoked by K+ in the absence of NT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Beta adrenoceptor facilitation of norepinephrine release is not dependent on local angiotensin II formation in the rat isolated kidney

The aim of the study was to investigate beta adrenoceptor modulation of norepinephrine release from sympathetic nerves in rat isolated kidney. After preincubation with [3H]norepinephrine, the renal nerves were stimulated at 1 Hz. The stimulation induced (S-I) outflow of radioactivity was taken as an index of norepinephrine release. Isoproterenol (0.1 microM) enhanced the S-I outflow of radioactivity. This effect was abolished by the beta-2 adrenoceptor blocking drug ICI 118551 (0.1 microM) but unaltered by the beta-1 adrenoceptor blocking drug atenolol (0.3 microM). In the presence of a high concentration of the angiotensin converting enzyme inhibitor captopril (5 microM), isoproterenol failed to enhance the S-I outflow of radioactivity. However, a lower concentration of captopril (0.1 microM), which totally abolished the facilitatory effect of angiotensin I (0.1 microM) on the S-I outflow of radioactivity, failed to alter the facilitatory effect of isoproterenol. Angiotensin II (0.03 microM) enhanced markedly the S-I outflow of radioactivity and in the presence of the angiotensin II receptor blocking drug saralasin (0.1 microM) this facilitatory effect was reduced markedly. Saralasin did not alter the facilitatory effect of isoproterenol. These results suggest that stimulation of prejunctional beta-2 adrenoceptors on renal sympathetic nerve endings enhances norepinephrine release. This effect is independent of local angiotensin II production and does not involve activation of prejunctional angiotensin II receptors within the rat kidney. However, the inhibitory effect of a high concentration of captopril (5.0 microM) on beta-2 adrenoceptor-mediated facilitation of norepinephrine release remains to be clarified.     

Rapid desensitization of dopamine release induced by neurotensin and neurotensin fragments

Neurotensin (NT), a tridecapeptide, induced a concentration-dependent release of dopamine (DA) from the striatum. In addition, NT8-13 and Nacetyl-NT8-13, the carboxy-terminal-containing hexapeptides, were much more effective as DA releasers than the amino-terminal, NT1-6 peptide. The potency and efficacy of NT in inducing DA release was markedly enhanced by increases in the extracellular concentration of potassium (K+). Similar to electrical stimulation and to elevated extracellular K+, NT-induced DA release was inhibited by 70% in the presence of 0.13 mM calcium. Desensitization to NT was observed after a first exposure to NT for 2.5 to 10 min, despite a 20- to 85-min washout period between exposures, with NT-free medium. The loss of response was not due to degradation or inactivation of the peptide, nor it was due to activation of DA autoreceptors or the DA transporter. NT-induced desensitization was not associated to a loss of responsiveness to DA release elicited by electrical stimulation or by high K+. In addition, desensitization occurred even if NT-induced DA release was markedly enhanced by high extracellular K+ (10 and 15 mM). Inhibition of NT-induced DA release by low calcium (on the first exposure) did not prevent the development of desensitization. Similar to the parent peptide, desensitization was observed with the active carboxy-terminal NT fragments. However, a first exposure to NT1-6 did not induce desensitization to NT8-13. These results are compatible with the view that NT-induced DA release and the development of desensitization are mediated through an action of NT on NT receptors.(ABSTRACT TRUNCATED AT 250 WORDS)