Tachykinin receptor antagonists inhibit hyperpnea-induced bronchoconstriction in guinea pigs.

We tested the hypothesis that hyperpnea-induced bronchoconstriction (HIB) and hyperpnea-induced bronchovascular hyperpermeability (HIBVH) are mediated through stimulation of NK-1 and NK-2 receptors in guinea pigs. We first established the efficacy and selectivity of (+/-) CP-96,345 (3 mg/kg i.v.) and of SR-48,968 (300 micrograms/kg i.v.) as NK-1 and NK-2 antagonists, respectively. (+/-) CP-96,345 substantially attenuated bronchoconstriction and systemic vascular leak caused by administration of Sar9,Met(O2)11-Substance P (a specific NK-1 agonist), but had no effect upon bronchoconstriction induced by selective NK-2 stimulation with Nle10-Neurokinin A[4-10]. Conversely, SR-48,968 antagonized the bronchoconstrictor response to Nle10-NKA[4-10], right-shifting the dose-response curve by 2 log units, but had no effect on Sar9, Met(O2)11-SP-induced bronchoconstriction. Anesthetized, tracheostomized, opened-chest male Hartley guinea pigs were pretreated with (+/-) CP-96,345 (3 mg/kg i.v.), SR-48,968 (300 micrograms/kg i.v.), or their respective vehicles, and Evans blue dye (30 mg/kg i.v.) to label circulating albumin. 10 min isocapnic dry gas hyperpnea (12 ml/kg, 150 breaths/min) provoked HIB and HIBVH in vehicle-treated animals. (+/-) CP-96,345 reduced the magnitude of HIB by one-half (peak posthyperpnea RL 7.8 +/- 1.9 [SE] times prehyperpnea baseline versus 16.1 +/- 2.6, vehicle-treated; P < or = 0.0001, ANOVA); SR-48,968 blocked HIB more completely (peak posthyperpnea RL 5.1 +/- 1.7 [SE] times prehyperpnea baseline versus 19.3 +/- 2.8, vehicle-treated; P < 0.0001, ANOVA). Neither drug reduced HIBVH. We conclude that dry gas hyperpnea causes bronchoconstriction in guinea pigs through activation of tachykinin receptors. The differential effects of neurokinin receptor blockade on HIB and HIBVH demonstrate that hyperpnea-induced airflow obstruction is not primarily a consequence of hyperpnea-induced bronchovascular leak.     

Tachykinins in the rat substantia nigra: effects of selective dopamine receptor antagonists

The effects of sustained blockade of dopamine receptors by selective dopamine antagonists on the tachykinin (substance P and neurokinin A) content in the substantia nigra were examined. The treatment of rats for 14 days with D-1/D-2 dopamine receptor antagonist haloperidol (2 mg/kg) or selective D-2 antagonist sulpiride (100 mg/kg) produced a similar and significant decrease in nigral substance P and neurokinin A-like immunoreactivity content, about 32-36% and 27-28% of control respectively. In contrast, administration of SCH 23390 (1 mg/kg), a selective and potent D-1 dopamine receptor antagonist, failed to affect the levels of substance P and neurokinin A in the substantia nigra and did not change the sulpiride-induced reduction of the nigral tachykinin peptides. These results indicate that the D-1 dopamine receptors are not involved in the modulation of nigral substance P and neurokinin A content and suggest that the blockade of the D-2 dopamine receptor subtype exerts the same regulation of the tachykinin gene expression, in spite of the existence of three mRNAs encoding substance P and neurokinin A.     

Involvement of alpha 7 nicotinic acetylcholine receptors in gene expression of dopamine biosynthetic enzymes in rat brain

Brain dopaminergic systems are critical in mediating the physiological responses to nicotine. The effects of several concentrations of nicotine (0.08, 0.17, or 0.33 mg/kg body weight) and involvement of alpha7 nicotinic acetylcholine receptors (nAChRs) in gene expression of key enzymes in dopamine biosynthesis were evaluated in the ventral tegmental area (VTA) and substantia nigra (SN), cell bodies of the mesocorticolimbic and nigrostriatal pathways. Nicotine elicited a dose-dependent elevation of mRNA for tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine biosynthesis in VTA and SN. The VTA was more sensitive to lower concentrations of nicotine with maximal response observed with the lowest dose of nicotine. Nicotine also elevated mRNA levels of GTP cyclohydrolase I (GTPCH), rate limiting in biosynthesis of TH's essential cofactor tetrahydrobiopterin in both dopaminergic locations. The changes in TH and GTPCH mRNAs were correlated. Pretreatment with the alpha7 nAChR antagonist methyllycaconitine prevented the nicotine-induced rise in TH or GTPCH mRNA in VTA and SN. Administration of alpha7 nAChR agonist 3-[2,4-dimethoxybenzilidene]anabaseine at 1 to 10 mg/kg or (E,E-3-(cinnamylidene)anabaseine at 0.3 to 1 mg/kg increased TH mRNA in VTA and SN, but not in peripheral catecholaminergic cells. Thus, agonists of alpha7 nAChRs have therapeutic potential for increasing TH gene expression in dopaminergic regions without some of nicotine's disadvantages, such as its harmful effects on the cardiovascular system. The findings indicate that nicotine may regulate dopamine biosynthesis by alterations in gene expression of TH and its cofactor. The alpha7 nAChRs are involved in mediating these effects of nicotine.     

In vivo dopamine clearance rate in rat striatum: regulation by extracellular dopamine concentration and dopamine transporter inhibitors

Dopamine transporter (DAT) inhibitors are expected to decrease dopamine (DA) clearance from the extracellular space of the brain. However, mazindol and cocaine have been reported to "anomalously" increase DA clearance rate. To better understand in vivo DAT activity both in the absence and presence of DAT inhibitors, clearance of exogenously applied DA was measured in dorsal striata of urethane-anesthetized rats using high-speed chronoamperometry. As higher amounts of DA were ejected, DA signal amplitudes, but not time courses, increased. Clearance rates increased until near maximal rates of 0.3 to 0.5 microM/s were attained. Provided baseline clearance rates were relatively low (< 0.1 microM/s), local application of either nomifensine or cocaine markedly increased exogenous DA signal amplitudes and time courses. Relative to the low baseline group, locally applied nomifensine decreased clearance rate when baseline clearance was high ( approximately 0.4 microM/s). However, even when baseline clearance rates were high, systemic injection of nomifensine, mazindol, GBR 12909, or benztropine increased DA signal amplitudes to a greater extent than time courses, consistent with the observed increases in clearance rates. In contrast, despite low baseline clearance rates, systemic injection of cocaine, WIN 35,428, or d-amphetamine preferentially increased DA signal time course, consistent with the observed decreases in clearance rates. Our results emphasize that as extracellular DA concentrations increase, DAT velocity increases to a maximum, partially explaining the ability of DAT inhibitors to increase DA clearance rates. However, by itself, kinetic activation is not sufficient to explain the ability of certain systemically administered DAT inhibitors to anomalously increase DA clearance.     

Nicotinic receptor modulation of dopamine transporter function in rat striatum and medial prefrontal cortex

Nicotine activates nicotinic acetylcholine receptors (nAChRs) on dopamine (DA) terminals to evoke DA release, which subsequently is taken back up into the terminal via the DA transporter (DAT). nAChRs may modulate DAT function thereby contributing to the regulation of synaptic DA concentrations. The present study determined the dose-response for nicotine (0.1-0.8 mg/kg, s.c.) to modulate DA clearance in striatum and medial prefrontal cortex (mPFC) using in vivo voltammetry in urethane anesthetized rats and determined if this effect was mediated by nAChRs. Exogenous DA (200 microM) was pressure-ejected at 5-min intervals until reproducible baseline signals were obtained. Subsequently, nicotine or saline was administered, and DA pressure ejection continued at 5-min intervals for 60 min. In both striatum and mPFC, signal amplitude decreased by approximately 20% across the 60-min session in saline-injected rats. A monophasic dose-response curve was found in striatum, with a maximal 50% decrease in signal amplitude after 0.8 mg/kg. In contrast, a U-shaped dose-response curve was found in mPFC, with a maximal 50% decrease in signal amplitude after 0.4 mg/kg. Onset of nicotine response occurred 10 to 15 min after injection in both brain regions; however, the amount of time before maximal response was 45 and 30 min in striatum and mPFC, respectively. Mecamylamine (1.5 mg/kg) completely inhibited the nicotine-induced (0.8 and 0.4 mg/kg) decrease in signal amplitude in striatum and mPFC, respectively, indicating mediation by nAChRs. Thus, nicotine enhances DA clearance in striatum and mPFC in a mecamylamine-sensitive manner, indicating that nAChRs modulate DAT function in these brain regions.     

A widespread abnormality of renin gene expression in the spontaneously hypertensive rat: modulation in some tissues with the development of hypertension

1. Renin messenger RNA (mRNA) levels were compared in the kidneys, livers, brains, adrenals, aortae and hearts of spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats at 5 and 12 weeks of age using a ribonuclease-protection technique. 2. Relative levels of renin mRNA were increased in the kidney, liver, brain, adrenal and heart of the young SHR compared with the WKY. In the aorta, levels were similar in the two strains at 5 weeks. 3. In 12-week-old animals, while increased levels persisted in the liver, brain and adrenal of the SHR, the level in the kidney was now the same in the two strains and the levels in the heart and aorta were lower in the SHR compared with the WKY. 4. Renin mRNA levels in the kidneys of SHR and WKY were also compared by Northern blotting and confirmed the observations made with the ribonuclease-protection technique. 5. The findings indicate a widespread abnormality of renin gene expression in the SHR which is modulated in some tissues by the development of hypertension. 6. While the mechanism(s) for the abnormality remains to be determined, the increased renin mRNA levels in the SHR in several tissues concerned with blood pressure regulation suggests an important role for the renin-angiotensin system in the development and maintenance of hypertension. 7. However, the finding of increased renin mRNA in the liver also suggests abnormalities in other, as yet unknown, functions of the renin-angiotensin system in the SHR.     

Primate trace amine receptor 1 modulation by the dopamine transporter

Recently identified trace amine receptors are potential direct targets for drugs of abuse, including amphetamine and 3,4-methylenedioxymethamphetamine (MDMA). We cloned full-length rhesus monkey trace amine receptor 1 (rhTA(1)) that was 96% homologous to human TA(1). The trace amines tyramine and beta-phenylethylamine (PEA) and the monoamine transporter substrates (+/-)-amphetamine and (+/-)-MDMA stimulated cAMP accumulation in rhTA(1)-expressing cell lines, as measured by a cAMP response element-luciferase assay. Cocaine did not stimulate cAMP accumulation in rhTA(1) cells, but it blocked [(3)H]PEA transport mediated by the dopamine transporter. Cotransfection with the human dopamine transporter enhanced PEA-, amphetamine-, and MDMA-mediated rhTA(1) receptor activation, but it diminished tyramine activation of rhTA(1). Because TA(1) (EGFP-rhTA(1) chimera) was largely intracellular, conceivably the dopamine transporter can facilitate access of specific agonists to intracellular TA(1). rhTA(1) mRNA expression was detected in rhesus monkey substantia nigra, implying that TA(1) may be colocalized with the dopamine transporter in dopamine neurons. In summary, primate TA(1) receptors are direct targets of trace amines, amphetamine, and MDMA. These receptors could also be indirect targets of amphetamine, MDMA, and cocaine through modification of monoamine transporter function. Conceivably, rhTA(1) receptors may be located on pre- or postsynaptic membranes. Interference with the carrier function of monoamine transporters with a consequent rise of extracellular levels of trace amines could activate these receptors. The cloning of a highly homologous TA(1) from rhesus monkey and demonstration that rhTA(1) receptors are activated by drugs of abuse, indicate that nonhuman primates may serve to model physiological and pharmacological TA(1)-mediated responses in humans.     

Cationic modulation of human dopamine transporter: dopamine uptake and inhibition of uptake.

Effects of cations on dopamine (DA) uptake into cells expressing the human dopamine transporter and on inhibition of DA uptake by various substrates and inhibitors were investigated by using rotating disk electrode voltammetry. The Na(+) dependence of DA uptake varied with Na(+) substitutes, hyperbolic with Li(+), almost linear at 1 microM DA but hyperbolic at 8 microM DA with choline, and sigmoidal with K(+). With Na(+) substituted by Li(+), K([DA]) decreased and V(app) remained constant with increasing [Na(+)], whereas K([Na+]) decreased and V(app) increased with increasing [DA], suggesting an ordered sequence with Na(+) binding before DA. Similar trends for the Na(+)-DA interactions were observed in the presence of cocaine. Cocaine inhibited DA uptake solely by increasing K([DA]), with its K(i) not significantly different at 55 and 155 mM [Na(+)], whereas it inhibited Na(+) stimulation by reducing V(app) more than K([Na+]) at 1 microM DA, and V(app) only and less potently at 8 microM DA. Thus, cocaine may compete with DA, not with Na(+), for the transporter, and might not follow a strictly ordered reaction with Na(+). With Na(+) substituted by K(+), K([DA]) or K([Na+]) became insensitive to Na(+) or DA. K(+) impaired the DA uptake mainly by reducing V(app,) but affected cocaine inhibition by elevating K(i). Despite their different patterns for inhibiting DA uptake, nontransportable inhibitors cocaine, methylphenidate, mazindol, and 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenyl-2-propyl)piperazi ne (GBR12909) showed similarly modest Na(+) dependence in their K(i) values. In contrast, substrates DA, m-tyramine, and amphetamine displayed a similarly stronger Na(+) requirement for their apparent affinities.     

Temperature-dependent modulation of [3H]nitrendipine binding by the calcium channel antagonists verapamil and diltiazem in rat brain synaptosomes

Binding of the dihydropyridine calcium channel antagonist [3H]nitrendipine to an intact rat brain mitochondrial-synaptosomal fraction (P2) was specific, saturable, temperature-dependent and of high affinity (Kd = 115-467 pM). The effects of the calcium channel antagonists verapamil and diltiazem on [3H]nitrendipine binding and their temperature dependence were investigated. At 0 and 25 degrees C, verapamil inhibited [3H]nitrendipine binding incompletely in a manner consistent with an allosteric modulation and nearly independent of the incubation temperature. The effects of diltiazem, however, were found to be highly temperature-dependent. At 25 and 37 degrees C, 10 microM diltiazem enhanced [3H]nitrendipine binding to values of 140 and 200% of control, respectively. At 0 degrees C, 10 microM diltiazem inhibited [3H]nitrendipine binding to a value of 68% of control. Analysis of saturation isotherms at steady state demonstrated that at all temperatures studied the effects of verapamil and diltiazem on [3H]nitrendipine binding were due to alterations in the ligand dissociation constant (Kd). At 25 degrees C, these alterations were mediated by changes in the rate of ligand-receptor complex dissociation. Competition studies of verapamil and diltiazem at 25 and 0 degrees C indicate that the effects of these two drugs on [3H]nitrendipine binding are mutually exclusive. We conclude that the binding of [3H]nitrendipine is allosterically modulated by spacially related binding sites for verapamil and diltiazem.     

Adeno-associated virus-mediated gene transfer to the brain: duration and modulation of expression

Adeno-associated virus (AAV) is a promising vector for central nervous system (CNS) gene transfer, but a number of issues must be addressed if AAV is to be used for widespread delivery throughout the CNS. Our aim was to test the effect of dose, route of delivery, and hydroxyurea treatment on brain expression of beta-galactosidase activity after cerebral inoculation with an rAAV-lacZ vector (rAAV-beta-gal). We also wished to test whether an immune response appeared against the vector and the transgene product. We found in BALB/c mice that beta-Gal expression increased during the first 2 months after inoculation, then decreased slightly by 4 months, and continued out to 6, 12, and 15 months in single animals. Cerebral injection produced localized beta-Gal expression that did not diffuse to other regions despite a fivefold increase in injection volume. Intraventricular injection resulted in negligible transduction. Antibodies to AAV capsid protein and beta-Gal appeared at low levels at 2 and 4 months, but correlated poorly with beta-Gal expression and did not prevent readministration of rAAV-beta-gal. Hydroxyurea treatment did not result in increased transduction in vivo. We conclude that our study confirms rAAV vectors as having considerable potential for CNS gene transfer; however, several important problems must be addressed if this vector system is to be used for long-term transduction of the entire brain. Sustained, regulatable expression will be needed if rAAV is to be used in the treatment of chronic CNS disease. The difficulty in delivering AAV to diverse regions of the brain is an important problem that must be overcome if these vectors are to be used for anything beyond localized transduction.     

Variation in the dopamine D2 receptor gene plays a key role in human pain and its modulation by transcranial magnetic stimulation

We tested whether variation of the dopamine D2 receptor (DRD2) gene contributes to individual differences in thermal pain sensitivity and analgesic efficacy of repetitive transcranial magnetic stimulation (rTMS) in healthy subjects (n = 29) or susceptibility to neuropathic pain in patients with neurophysiologically confirmed diagnosis (n = 16). Thermal sensitivity of healthy subjects was assessed before and after navigated rTMS provided to the S1/M1 cortex. All subjects were genotyped for the DRD2 gene 957C>T and catechol-O-methyltransferase (COMT) protein Val158Met polymorphisms. In healthy subjects, 957C>T influenced both innocuous and noxious thermal detection thresholds that were lowest in 957TT homozygotes (P values from .0277 to .0462). rTMS to S1 cortex had analgesic effect only in 957TT homozygote genotype (P = .0086). In patients, prevalence of 957TT homozygote genotype was higher than in a healthy Finnish population (50% vs 27%; P = .0191). Patients with 957TT genotype reported more severe pain than patients with other genotypes (P = .0351). COMT Val158Met polymorphism was not independently associated with the studied variables. Genetic regulation of DRD2 function by 957C>T polymorphism thus seems to influence thermal and pain sensitivity, its modulation by rTMS, and susceptibility to neuropathic pain. This indicates a central role for the dopamine system and DRD2 in pain and analgesia. This may have clinical implications regarding individualized selection of patients for rTMS treatment and assessment of risks for neuropathic pain.     

Differential regulation of the endocannabinoids anandamide and 2-arachidonylglycerol within the limbic forebrain by dopamine receptor activity

Glutamatergic synaptic transmission within the striatum and prefrontal cortex regulates the neuronal synthesis of endocannabinoids. Because a primary role of dopamine is to modulate this excitatory transmission, we tested the hypothesis that dopaminergic transmission modulates endocannabinoid content in the limbic forebrain. Liquid chromatography/mass spectrometry was used to determine endogenous anandamide and 2-arachidonylglycerol (2-AG) contents within the limbic forebrain of mice after pharmacological manipulation of dopaminergic transmission. Increasing synaptic dopamine concentrations with methylphenidate significantly and dose dependently decreased both anandamide and 2-AG content. The selective dopamine reuptake inhibitor 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine (GBR 12909) also significantly decreased anandamide and tended to decrease 2-AG content. The D1 receptor antagonist R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH 23390) increased and the D1 receptor agonist 2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine (SKF 33939) decreased anandamide content. 2-AG content was unaffected by SCH 23390 but was significantly increased by the D2 receptor antagonist eticlopride, which had no effect on anandamide content. The D2 agonist quinpirole had a biphasic effect on anandamide content with low, autoreceptor-preferring doses increasing anandamide and higher doses decreasing it back toward control. Quinpirole did not significantly affect 2-AG content. Together, these data indicate that endogenous dopamine exerts a differential, net suppressive effect upon anandamide and 2-AG content via activation of D1 and D2 receptors, respectively. These data are consistent with the hypothesis that modulation of endocannabinoid content by dopamine is secondary to changes in glutamatergic transmission, and they provide a pharmacological framework for the rational development of endocannabinoid-based therapeutic interventions for dopamine-related neuropsychiatric disorders.     

Nicotinic modulation of mesoprefrontal dopamine neurons: pharmacologic and neuroanatomic characterization

Schizophrenics have cortical dysfunction that may involve mesoprefrontal dopamine (DA) systems. Rates of nicotine dependence approach 90% in schizophrenia, and nicotine administration through cigarette smoking may ameliorate cognitive dysfunction, which may be related to cortical DA dysregulation. We have shown that repeated, but not acute, nicotine pretreatment (0.15 mg/kg daily s.c.) reduces footshock stress-induced mesoprefrontal DA metabolism and immobility responses. This effect of repeated nicotine is dependent on mecamylamine (MEC)-sensitive nicotinic acetylcholine receptor (nAChR) stimulation and endogenous opioid peptides. In the present study, we have further characterized these effects of repeated nicotine on the stress reactivity of mesoprefrontal DA neurons by using the following: 1) local infusion of MEC into cell bodies (ventral tegmental area) and terminal fields (medial prefrontal cortex) to determine the site of action of nicotine; and 2) systemic administration of selective nAChR antagonists. Results of bilateral local infusions of MEC (0.1-1.0 microgram/side) into ventral tegmental area or medial prefrontal cortex in saline- and nicotine-pretreated rats suggests a modulatory role for somatodendritic versus terminal field nAChRs on mesoprefrontal DA neurons under stress-induced states. Experiments with dihydro-beta-erythroidine (a beta2-subunit-selective blocker; 0.0-3.0 mg/kg) and methylycaconitine (an alpha7-subunit-selective blocker; 0.0-8.4 mg/kg) suggest that both alpha4beta2- and alpha7-containing nAChRs modulate mesoprefrontal DA neurons. Thus, complex regulation of mesoprefrontal DA neurons by nAChRs is suggested, which may have relevance to prefrontal cortical DA dysfunction and the high comorbid rates of nicotine dependence in schizophrenia.