Levodopa dose and severity of Parkinson’s disease (PD) are recognized risk factors for levodopa-induced dyskinesia (LID) in humans. The purpose of the present study was to evaluate the ability of these variables to predict severity of LID in a rat model of PD. Varied concentrations of 6-hydroxy-dopamine were injected into the midbrain to produce wide ranges of dopamine depletion in striatum. Three weeks later, rats were given daily injections of levodopa (2–10 mg/kg i.p.) plus benserazide (12.5 mg/kg i.p.) for 15 days. Abnormal involuntary movements (AIMs) were measured for limb, axial, orolingual, and rotatory movements. Dose-response analysis for total AIM scores yielded a levodopa ED50 value of 3.2 mg/kg on treatment day 15. There were strong interrelated correlations between individual AIM categories (ρ > 0.7) and for each AIM category in regard to total AIM score (ρ > 0.7). In rats that received levodopa doses that were greater than the ED50, rates of amphetamine-induced rotation were significantly correlated with total AIM scores (ρ = 0.413). However, of those rotating >5 times/min, 34% had relatively low AIM scores (<8). Likewise, there was a significant correlation between percentages of tyrosine hydroxylase (TH) loss and total AIM scores (ρ = 0.388). However, in those rats that had >85% TH loss, 30% had AIM scores <8. Our results show that given an adequate dose and magnitude of striatal dopamine depletion, levodopa produces dyskinesia with a continuous spectrum of severity. Although levodopa dose and level of dopamine depletion are significant risk factors for LID, we conclude that other factors must contribute to LID susceptibility.

Prostaglandin E2 (PGE2), the most relevant eicosanoid promoting inflammation and tumorigenesis, is formed by cyclooxygenases (COXs) and PGE2 synthases from free arachidonic acid. Preparations of the leaves of Salvia officinalis are commonly used in folk medicine as an effective antiseptic and anti-inflammatory remedy and possess anticancer activity. Here, we demonstrate that a standard ethyl acetate extract of S. officinalis efficiently suppresses the formation of PGE2 in a cell-free assay by direct interference with microsomal PGE2 synthase (mPGES)-1. Bioactivity-guided fractionation of the extract yielded closely related fractions that potently suppressed mPGES-1 with IC50 values between 1.9 and 3.5 μg/ml. Component analysis of these fractions revealed the diterpenes carnosol and carnosic acid as potential bioactive principles inhibiting mPGES-1 activity with IC50 values of 5.0 μM. Using a human whole-blood assay as a robust cell-based model, carnosic acid, but not carnosol, blocked PGE2 generation upon stimulation with lipopolysaccharide (IC50 = 9.3 μM). Carnosic acid neither inhibited the concomitant biosynthesis of other prostanoids [6-keto PGF1α, 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid, and thromboxane B2] in human whole blood nor affected the activities of COX-1/2 in a cell-free assay. Together, S. officinalis extracts and its ingredients carnosol and carnosic acid inhibit PGE2 formation by selectively targeting mPGES-1. We conclude that the inhibitory effect of carnosic acid on PGE2 formation, observed in the physiologically relevant whole-blood model, may critically contribute to the anti-inflammatory and anticarcinogenic properties of S. officinalis.

Tissue injury in mammals triggers both inflammatory and repair responses that, in some contexts, results in fibrosis. Fibrosis is characterized by the persistence of activated myofibroblasts, ineffective re-epithelialization, and variable degrees of inflammation within injured tissues. The protein kinase inhibitor (PKI), imatinib mesylate, has been proposed as a potential antifibrotic therapeutic agent. In this study, the efficacy of imatinib mesylate to modulate fibrogenic responses, both in vitro and in vivo, was examined. In an in vitro fibroblast culture model, imatinib inhibits platelet-derived growth factor receptor activation and fibroblast proliferation but not the stably differentiated myofibroblast phenotype. Furthermore, imatinib inhibits lung epithelial cell proliferation and survival but not the induction of epithelial-mesenchymal transition. Imatinib does not alter transforming growth factor-β/SMAD3 signaling in either cell type. In a murine model of lung fibrosis, bleomycin-induced injury to the pulmonary epithelium provokes an early inflammatory response with more delayed fibrosis during the late reparative phase of lung injury. Imatinib mesylate (10 mg/kg/day by i.p. injection or oral gavage), administered during the postinjury repair phase, failed to significantly alter fibrogenic responses assessed by histopathology, collagen content, and the accumulation of myofibroblasts within the injured lung. These studies indicate that the capacity of a PKI to inhibit fibroblast proliferation may be insufficient to mediate significant antifibrotic effects in late stages of tissue injury repair. Pharmacologic agents that modulate the activities and fate of differentiated (myo)fibroblasts, without interfering with the regenerative capacity of epithelial cells, are likely to be more effective for treatment of nonresolving, progressive fibrotic disorders.

The human multidrug resistance gene MDR1 encodes a membrane-bound transporter P-glycoprotein (Pgp) that confers the drug resistance of cancer cells by mediating an ATP-dependent drug efflux transport. We and others have reported a number of functionally significant MDR1 variants, including G1199A and G1199T, that modulate cancer drug resistance and intracellular levels of antivirals. In this report, we describe a novel G571A variant of MDR1 detected in 6.4% of leukemia patients. Because this nucleotide modification gives rise to an amino acid change from Gly to Arg at the 191 amino acid position of Pgp, we have developed and characterized the functional affect of the G571A variant in stable, recombinant cells. Using six chemotherapeutic drugs, doxorubicin HCl, daunorubicin HCl, vinblastine sulfate, vincristine sulfate, taxanes (paclitaxel), and epipodophyllotoxin (etoposide, VP-16), we found that the MDR1571A variant selectively reduced the degree of Pgp-mediated resistance in drug-dependent manner. Although there was a minimal effect on doxorubicin and daunorubicin, the MDR1-dependent resistance on vinblastine, vincristine, paclitaxel, and etoposide was reduced by approximately 5-fold. The increased drug sensitivity in MDR1571A, compared with MDR1wt, paralleled the intracellular drug levels. These data suggest that individuals with this novel MDR1 variant, the 571A genotype, may be more sensitive to the specific anticancer drugs that are Pgp substrates.

XAC (xanthine amine congener, 8-{4-[(2-aminoethyl)-aminocarbonylmethyloxy]phenyl}-1,3-dipropylxanthine is a potent adenosine antagonist that reverses the reduction in urine flow, sodium excretion and heart rate produced by the adenosine agonist, N6-cyclohexyladenosine. New derivatives of XAC in which the primary amino group has been condensed to the γ-carboxyl group of glutamic acid have been synthesized as prodrugs. These amino acid-XAC conjugates, which are considerably less potent than XAC in competitive binding assays at A1-adenosine receptors, are designed for selective enzymatic activation in the kidneys. The γ-glutamyl xanthine derivatives are substrates for γ-glutamyl transferase (EC 2.3.2.2) to generate an amine-functionalized xanthine. N-acetyl-γ-L-glutamyl-XAC is not active in vivo, consistent with inability of renal acylase (EC 3.5.1.14) to hydrolyze the acetyl group, a prerequisite step for the production of XAC from this molecule. The xanthine derivatives, γ-L-glutamyl-XAC and γ-L-glutamyl-γ-L-glutamyl-XAC are metabolized to XAC and produce a diuresis in vivo.

The platelet P2Y1 ADP receptor is an attractive target for new antiplatelet drugs. However, because of the lack of strong and stable antagonists, only a few studies have suggested that pharmacological inhibition of the P2Y1 receptor could efficiently inhibit experimental thrombosis in vivo. Our aim was to determine whether the newly described potent and selective P2Y1 receptor antagonist MRS2500 [2-iodo-N6-methyl-(N)-methanocarba-2′-deoxyadenosine-3′,5′-bisphosphate] could inhibit platelet function ex vivo and experimental thrombosis in mice in vivo. MRS2500 was injected intravenously into mice, and its effect on ex vivo platelet aggregation and in several models of thrombosis in vivo was determined. MRS2500 displayed high potency and stable and selective P2Y1 receptor inhibition ex vivo. Although MRS2500 injection resulted in only moderate prolongation of the bleeding time, it provided strong protection in systemic thromboembolism induced by infusion of a mixture of collagen and adrenaline. MRS2500 also potently inhibited localized arterial thrombosis in a model of laser-induced vessel wall injury with two degrees of severity. Moreover, combination of MRS2500 with clopidogrel, the irreversible inhibitor of the platelet P2Y12 receptor for ADP, led to increased antithrombotic efficacy compared with each alone. These results add further evidence for a role of the P2Y1 receptor in thrombosis and validate the concept that targeting the P2Y1 receptor could be a relevant alternative or complement to current antiplatelet strategies.

ADP is the cognate agonist of the P2Y1, P2Y12, and P2Y13 receptors. With the goal of identifying a high potency agonist that selectively activates the P2Y1 receptor, we examined the pharmacological selectivity of the conformationally constrained non-nucleotide analog (N)-methanocarba-2MeSADP [(1′S,2′R, 3′S,4′R,5′S)-4-[(6-amino-2-methylthio-9H-purin-9-yl)-1-diphosphoryloxymethyl]bicyclo[3.1.0]hexane-2,3-diol] among the three ADP-activated receptors. Each P2Y receptor was expressed transiently in COS-7 cells, and inositol lipid hydrolysis was quantified as a measure of receptor activity. In the case of the Gi-linked P2Y12 and P2Y13 receptors, a chimeric G protein, Gαq/i, was coexpressed to confer a capacity of these Gi-linked receptors to activate phospholipase C. 2MeSADP (2-methylthio-ADP) was a potent agonist at all three receptors exhibiting EC50 values in the sub to low nanomolar range. In contrast, whereas (N)-methanocarba-2MeSADP was an extremely potent (EC50 = 1.2 ± 0.2 nM) agonist at the P2Y1 receptor, this non-nucleotide analog exhibited no agonist activity at the P2Y12 receptor and very low activity at the P2Y13 receptor. (N)-Methanocarba-2MeSADP also failed to block the action of 2MeSADP at the P2Y12 and P2Y13 receptors, indicating that the (N)-methanocarba analog is not an antagonist at these receptors. The P2Y1 receptor selectivity of (N)-methanocarba-2MeSADP was confirmed in human platelets where it induced the shape change promoted by P2Y1 receptor activation without inducing the sustained platelet aggregation that requires simultaneous activation of the P2Y12 receptor. These results provide the first demonstration of a high-affinity agonist that discriminates among the three ADP-activated P2Y receptors, and therefore, introduce a potentially important new pharmacological tool for delineation of the relative biological action of these three signaling proteins.

Manganese (Mn) exposure causes Manganism, a neurological disorder similar to Parkinson’s disease. However, the cellular mechanism by which Mn induces dopaminergic neuronal cell death remains unclear. In the present study, we sought to investigate the key downstream apoptotic cell signaling events that contribute to Mn-induced cell death in mesencephalic dopaminergic neuronal (N27) cells. Mn exposure induced a dose-dependent increase in neuronal cell death in N27 cells. The cell death was accompanied by sequential activation of mitochondrial-dependent proapoptotic events including cytochrome c release, caspase-3 activation, and DNA fragmentation, but not caspase-8 activation, indicating that the mitochondrial-dependent apoptotic cascade primarily triggers Mn-induced apoptosis. Notably, Mn treatment proteolytically activated protein kinase Cδ (PKCδ), a member of a novel class of protein kinase C. The caspase-3 specific inhibitor Z-DEVD-FMK significantly blocked PKCδ cleavage and its kinase activity, indicating that caspase-3 mediates the proteolytic activation. Co-treatment with the PKCδ inhibitor rottlerin or the caspase-3 inhibitor Z-DEVD-FMK almost completely blocked Mn-induced DNA fragmentation. Additionally, N27 cells expressing a catalytically inactive PKCδK376R protein (PKCδ dominant negative mutant) or a caspase cleavage resistant PKCδD327A protein (PKCδ cleavage resistant mutant) were found to be resistant to Mn-induced apoptosis. To further establish the proapoptotic role of PKCδ, RNAi-mediated gene knockdown was performed. siRNA suppression of PKCδ expression protected N27 cells from Mn-induced apoptotic cell death. Collectively, these results suggest that caspase-3-dependent proteolytic activation of PKCδ plays a key role in Mn-induced apoptotic cell death.

The locomotor effects in mice of selective A1 and A2 adenosine agonists, antagonists and combinations of agonists were investigated using a computerized activity monitor. The A2-selective agonist 2-[(2-aminoethylamino)carbonylethylphenylethylamino]-5'-N-ethylcarboxamidoadenosine (APEC), an amine derivative of 2-(carboxyethylphenylethylamino)adenosine-5'-carboxamide, was a more potent locomotor depressant than its amide conjugates. The rank order of potency after i.p. injection for adenosine agonists was 5'-N-ethylcarboxamidoadenosine (NECA) (ED50, 5.8 nmol/kg) > APEC (ED50, 25 nmol/kg) > N6-cyclohexyladenosine (CHA) (ED50, 270 nmol/kg). An A1-selective, centrally acting, adenosine antagonist, 8-cyclopentyltheophylline (10 mg/kg), completely reversed the locomotor depressant effects of CHA (A1-selective) and NECA (nonselective) at doses of agonists as high as twice the ED50, and shifted the dose-response curves to the right, suggesting a primary involvement of A1 receptors. 8-cyclopentyltheophylline did not affect the depressant effects of APEC at the ED50, consistent with the A2-selectivity of APEC. The locomotor effects of APEC and CHA were completely reversed by theophylline, but not by the peripherally active 8-p-sulfophenyltheophylline, indicating central action of the adenosine agonists. The depressant effects of APEC, but not of NECA or CHA, were reversed significantly by an A2-selective adenosine receptor antagonist, 4-amino-8-chloro-1-phenyl-[1,2,4]triazol[4,3-a]quinoxaline. Low or subthreshold doses of CHA potentiated the depressant effects of APEC. A subthreshold dose of CHA did not alter the depressant effect of NECA, whereas a subthreshold dose of APEC increased the depressant effects of low doses of NECA. Thus, it appears that A1- and A2-selective adenosine agonists have separate central depressant effects, which can be potentiative. The relatively high potency of NECA in vivo could be due to a synergism between central A1 and A2receptor activation by this nonselective agonist.

L-Glutamate (Glu) is the main excitatory neurotransmitter in the mammalian central nervous system, and it is involved in most aspects of normal brain function, including cognition, memory and learning, plasticity, and motor movement. Although micro-dialysis techniques have been used to study Glu, the slow temporal resolution of the technique may be inadequate to properly examine tonic and phasic Glu. Thus, our laboratory has developed an enzyme-based microelectrode array (MEA) with fast response time and low detection limits for Glu. We have modified the MEA design to allow for reliable measures in the brain of awake, freely moving mice. In this study, we chronically implanted the MEA in prefrontal cortex (PFC) or striatum (Str) of awake, freely moving C57BL/6 mice. We successfully measured Glu levels 7 days postimplantation without loss of MEA sensitivity. In addition, we determined resting (tonic) Glu levels to be 3.3 μM in the PFC and 5.0 μM in the Str. Resting Glu levels were subjected to pharmacological manipulation with tetrodotoxin (TTX) and DL-threo-β-hydroxyaspartate (THA). TTX significantly (p < 0.05) decreased resting Glu by 20%, whereas THA significantly (p < 0.05) increased resting Glu by 60%. Taken together, our data show that chronic recordings of tonic and phasic clearance of exogenously applied Glu can be carried out in awake mice for at least 7 days in vivo, allowing for longer term studies of Glu regulation.

Inhibition of the sodium-proton exchanger (NHE) plays an important role in reducing tissue damage during ischemic reperfusion injury; however, pharmacological inhibitors of NHE have restricted access to acutely ischemic tissues because of severely compromised tissue perfusion. We describe the syntheses, characterization, and NHE inhibitory activities of a novel class of amiloride derivatives where peptides are conjugated to the amiloride C(5) amino group. These new peptide-C(5)-amiloride conjugates are inactive; however, peptide residues were chosen such that selective cleavage by neutral endopeptidase 24.11 (enkephalinase) liberates an amino acid-C(5)-amiloride conjugate that inhibits NHE in a glial cell line. These results confirm the feasibility of using peptide-amiloride conjugates as NHE inhibitor prodrugs. We envision the design of analogous peptide-amiloride prodrugs that can be administered prior to ischemic events and subsequently activated by endopeptidases selectively expressed by ischemic tissues.

We previously reported that apolipoprotein (Apo) E-deficient, ApoB48-containing (E−/B48) lipoproteins inhibited expression of lysosomal hydrolase and transformed mouse peritoneal macrophages (MPMs) into foam cells. The present study examined the effect of 2-aminopurine (2-AP), an inhibitor of eukaryotic initiation factor (eIF)-2α phosphorylation, on E−/B48 lipoprotein-induced changes in gene expression and foam cell formation. Our data demonstrated that E−/B48 lipoproteins enhanced phosphorylation of eIF-2α in macrophages. Incubation of MPMs with E−/B48 lipoproteins inhibited the translation efficiency of mRNAs encoding lysosomal acid lipase, cathepsin B, and cation-dependent mannose 6 phosphate receptor, with a parallel reduction in the level of these proteins. Addition of 2-AP to the culture media alleviated the suppressive effect of E−/B48 lipoproteins on lysosomal hydrolase mRNA translation, increased macrophage degradation of E−/B48 lipoproteins, and inhibited foam cell formation. Transfection of MPMs with a nonphosphorylatable eIF-2α mutant also attenuated the suppressive effect of E−/B48 lipoproteins on expression of lysosomal acid lipase, associated with a reduced accumulation of cellular cholesterol esters. This is the first demonstration that ApoE-deficient lipoproteins inhibit lysosomal hydrolase synthesis and transform macrophages into foam cells through induction of eIF-2α phosphorylation.

Opiate dependence and withdrawal have long been hypothesized to enhance the reinforcing effects of opiates; however, opiate agonist self-administration in these states has yet to be systematically assessed. To address this issue, the reinforcing property of the short-acting μ-opioid agonist, remifentanil, was assessed in morphine-dependent (MD), morphine-dependent and -withdrawn (MW), and nondependent, control (C) rats. Dependence was established by twice daily administration of increasing doses of morphine for 4 days (10, 20, 30, and 40 mg/kg s.c.) and then maintained with a daily injection of the large dose. Morphine deprivation-induced withdrawal (defined by weight loss and hyperalgesia) was apparent 24, but not 12, h after morphine treatment. Remifentanil self-administration (0.4, 0.8, 1.6, 3.2, or 6.4 μg/kg/infusion) was assessed over 20 successive, daily, 1-h sessions, either 12 or 24 h after the maintenance dose of morphine. Compared with the control group, the MD group demonstrated suppressed remifentanil self-administration, whereas the MW group exhibited enhanced responding for every dose of remifentanil. The increased responding observed in the MW group compared with the control and MD groups resulted in an upward shift in the remifentanil dose-response curve, an effect that was expressed only after repeated exposure to the contingency, demonstrating that morphine withdrawal ultimately enhances the reinforcing effects of remifentanil.

The relative anticonvulsant potential of the γ-aminobutyric acid (GABA) agonist, muscimol, was compared after microinjection into either the inferior colliculus, substantia nigra or medial septum of ethanol-dependent rats. Bilateral microinjection of muscimol (10–30 ng) into the inferior colliculus 15 to 60 min before testing suppressed all sound-induced seizure components (wild running, clonus and tonus) in rats withdrawn from ethanol for 6.5 to 8.5 hr. However, forelimb tremors were not altered. Audiogenic seizures were suppressed for at least 3 hr after muscimol (30 ng). In the medial septum and substantia nigra, microinjection of muscimol (30–100 ng) only partially reduced the tonic component of audiogenic seizures and exerted no effect on the frequency of wild running or clonus. GABA (10 μg) and two other GABA agonists [4,5,6,7-tetrahydroisoxa-zolo[5,40c]pyridin-3-ol (THIP), 300 ng and chlordiazepoxide, 10–30 μg], microinjected into the inferior colliculus, also reduced audiogenic seizure susceptibility. However, 1,3-butanediol, which suppresses ethanol withdrawal seizures after peripheral administration in rats, was inactive. The relative proconvulsant potential of the GABA antagonist, bicuculline methiodide, also was compared after microinjection into either the inferior colliculus, substantia nigra or medial septum of ethanol naive rats. In each animal, audiogenic seizure-like wild running, clonus and tonus were evoked by microinjecting bicuculline methiodide into the inferior colliculus at the rate of 6.0 ng/6 min. However, these reactions did not occur when bicuculline methiodide was applied at a slower rate (1.8 ng/6 min). Similar injections of bicuculline methiodide (600 ng/6 min) into the substantia nigra caused only clonus and tonus without wild running. A smaller dose (180 ng/6 min) had no effect. In the medial septum, microinjection of this GABA antagonist (1800 ng/6 min) did not exert any obvious seizure-like activity. These results suggest that the inferior colliculus is important in GABAmimetic suppression of audiogenic seizures and that reduced GABAergic activity in this nucleus may be responsible for the increased susceptibility to audiogenic seizures in rats during ethanol withdrawal.

The α adrenergic receptor subtypes of the human prostate have been intensively investigated, while the muscarinic receptor subtypes and their function have yet to be determined in this tissue. [3H]-QNB binding to muscarinic receptors was performed on membrane homogenates of adenoma from six prostatectomy specimens resulting in an average total receptor density of 46 fMol/mg protein. Pirenzepine, hexahydrosiladifenidol, and para-fluoro-hexahydrosiladifenidol, drugs with high affinity for the M1 subtype, were significantly more potent inhibitors of [3H]-QNB binding than the M2 selective drug methoctramine. Immunoprecipitation studies were done using antisera raised to individual M1–M5 receptor subtypes. Approximately 75% of the solubilized receptors in the adenoma specimens were immunoprecipitated with the anti-M1 antibody, in contrast to 5% or less with antibodies against M2, M3 or M4 subtypes. These immunoprecipitation studies confirm the preponderance of the M1 subtype in prostate adenoma suggested by the high affinity pirenzepine binding. M1 receptors, when incubated with agonist, coimmunoprecipitated with the α subunits of the guanine nucleotide binding regulatory proteins Giα, Gq/11α and G16α. Immunohistochemical staining with the anti-M1 antibody demonstrates the M1 receptor to be localized to the glandular epithelium. The human prostate is the first peripheral tissue in which a preponderance of the M1 subtype of muscarinic receptors has been demonstrated.

The regulation of muscarinic acetyicholine receptor (MAChR) subtypes in rat striatum, bladder and heart was examined following a 14-day administration of neuroleptics (clozapine or fluphenazine), anticholinergics (atropine) or a combination of anticholinergics and neuroleptics. Levels of MAChRs were ascertained by the use of immunoprecipitation and radioligand binding. The combined treatment of fluphenazine and atropine produced an increase in all MAChR subtype levels in striatum with m1 receptor levels having the largest increase (270%) from control. A significant increase (105%) was also seen striatal in m2 receptor levels. Residual muscarinic receptor levels, representing the m3 and m4 subtypes, were increased (72%) to a lesser degree above control. Fluphenazine treatment alone increased levels of the m2 MAChR, whereas clozapine administration had no significant effect on levels of any MAChR subtype in this tissue. Administration of the cholinergic antagonist, atropine, showed a significant increase (89%) in the striatal m1 MAChR subtype. Of the MAChRs found in rat bladder and rat heart, the m2 subtype has been shown to be the most abundant. Results from the rat bladders indicated a reduction (50%) in muscarinic antagonist binding that was limited to the fluphenazine treatment group. In heart, atropine treatment alone produce a slight increase (ca. 10%) in receptor binding. No significant effect on muscarinic receptor levels was seen with the other treatment groups. These data demonstrate that there are differences in muscarinic receptor level modulation between central and peripheral tissues.

Muscarinic acetylcholine receptors, particularly M3 receptors, are physiologically the most important mechanism to induced urinary bladder smooth muscle contraction. Their prototypical signaling response is a stimulation of phospholipase C (PLC), and this also has been shown in the urinary bladder. Nevertheless, it has remained controversial whether PLC signaling mediates bladder contraction induced by muscarinic receptor agonists. Studies in favor and against a role for PLC differed in their experimental protocol (single versus repeated concentration-response curves within a single preparation) and in the PLC inhibitors that have been used. We have now tested whether previous differential conclusions regarding a role for PLC are related to inhibitors and/or experimental protocols. In a single curve protocol, U-73,122 [1-[6-[((17β)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione] did not attenuate carbachol responses. In a repeated curve protocol, ET-18-OCH3 (1-O-octa-decyl-2-O-methyl-sn-glycero-3-phosphorylcholine) lacked significant inhibition relative to vehicle time controls. In contrast, D609 (O-tricyclo[5.2.1.02,6]dec-9-yl dithiocarbonate potassium salt) depressed maximal carbachol effects but also nonspecifically inhibited contraction induced by KCl. Neomycin did not affect the carbachol-induced rat urinary bladder contraction. We conclude that previously reported differences relate to the use of inhibitors rather than experimental protocols and that the overall data do not support a role for PLC in M3 muscarinic receptor-mediated rat bladder contraction.

Activation of muscarinic acetylcholine receptors is primarily responsible for urinary bladder emptying. Because multiple subtypes of muscarinic receptors exist, we wished to characterize those present in bladder and ultimately to attribute function to those that regulate bladder contractility, neurotransmitter release and perhaps other cholinergic functions in this tissue. Although the m2 and m3 subtypes could be immunoprecipitated after solubilization from human, rat, rabbit and guinea pig bladder membranes, the m1, m4 and m5 subtypes could not. The m2:m3 ratio was 9:1 in rat bladder but was only 3:1 in the other species examined. Immunoprecipitation of the m2 subtype correlated with the relative levels of high-affinity agonist binding sites measured by competition of carbachol for [3H]N-methylscopolamine binding or measured directly using [3H]oxotremorine-M. In the presence of agonist, but not antagonist, GTP binding proteins could be immunoprecipitated in concert with the m2 or m3 receptors using anti-receptor antibodies. These proteins were members of the Gi and Gq/11 subfamilies for both the m2 and the m3 receptor subtypes. In spite of the preponderance of the m2 receptor in all species studied, Schild analysis using somewhat selective antagonists showed that the pharmacologically defined m3 receptor mediated contractility in strips of rat and rabbit bladder. Thus acetylcholine activates bladder smooth muscle via the m3 receptor subtype, and subsequent contractility may be transduced by guanine nucleotide binding proteins such as the Gi and Gq/11 subfamilies.

Smooth muscle contains multiple muscarinic receptor subtypes, including M2 and M3. M2 receptors outnumber M3 receptors. Based on the potency of subtype selective anticholinergics, contraction is mediated by the M3 subtype. However, results from knockout (KO) mice show that the M2 receptor mediates approximately 45% of the contractile response produced by the M3 receptor. The traditional theory of one receptor mediating a response does not allow assessment of interactions between receptors when more than one receptor participates in a response. Our study was performed using a novel analysis method based on dual receptor occupancy to determine how M2 and M3 receptor subtypes interact to mediate contraction in mouse stomach. Cumulative carbachol concentration contractile responses were determined for wild-type, M2-KO, and M3-KO stomach body smooth muscle. Using affinity constants for carbachol at M2 and M3 cholinergic receptors, the concentration values were converted to fractional receptor occupation. The resulting occupation-effect relations showed maximum effects for the M2 and M3 subtypes, respectively. These occupation-effect relations allow determination of the additive (expected) isobole based on this dual occupancy, thereby providing a curve (mathematically derived) for comparison against the experimentally derived value in wild type. The actual values determined experimentally in the wild type were not statistically significantly different from that predicted by the isobole. This confirms that the interaction between these mutually occupied receptors is additive. The new method of analysis also expands the traditional Schild theory that was based on a single receptor type to which the agonist and antagonist bind.

Normal rat bladder contractions are mediated by the M3 muscarinic receptor subtype. The M2 receptor subtype mediates contractions of the denervated, hypertrophied bladder. This study determined signal transduction mechanisms mediating contraction of the denervated rat bladder. Denervated bladder muscle strips were exposed to inhibitors of enzymes thought to be involved in signal transduction in vitro followed by a cumulative carbachol concentration-response curve. Outcome measures were the maximal contraction, the potency of carbachol, and the affinity of darifenacin for inhibition of contraction. Inhibition of phosphoinositide-specific phospholipase C (PI-PLC) with 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphorylcholine (ET-18-OCH3) has no effect on denervated bladder contractions, whereas inhibition of phosphatidyl choline-specific phospholipase C (PC-PLC) with O-tricyclo[5.2.1.02,6]dec-9-yl dithiocarbonate potassium salt (D609) attenuates the carbachol maximum and potency. Inhibition of rho kinase with (R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride (Y-27632) reduces carbachol maximum, carbachol potency, and increases darifenacin affinity. Inhibition of rho kinase, protein kinase A (PKA), and protein kinase G (PKG) with 1-(5-isoquinolinesulfonyl)-homopiperazine·HCl (HA-1077) reduces the carbachol maximum and potency. Inhibition of PKC with chelerythrine increases darifenacin affinity, whereas inhibition of rho kinase, PKA, PKG, and protein kinase C (PKC) with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine·2HCl (H7) reduces the carbachol potency while increasing darifenacin affinity. Inhibition of rho kinase, PKA, and PKG with N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide·2HCl (H89) increases darifenacin affinity. This study demonstrates that different signal transduction mechanisms mediate the contractile response in the denervated rat bladder than in normal rat bladder. In normal rat bladder, PI-PLC and PC-PLC mediate the contraction, but in denervated bladder only PC-PLC is involved. In the denervated bladder, the rho kinase pathway is more dominant than in normal bladders. PKA seems to mediate a contractile response in normal bladders, whereas it seems to inhibit contraction in denervated bladders.

The muscarinic receptor subtype-activated signal transduction mechanisms mediating rat urinary bladder contraction are incompletely understood. M3 mediates normal rat bladder contractions; however, the M2 receptor subtype has a more dominant role in contractions of the hypertrophied bladder. Normal bladder muscle strips were exposed to inhibitors of enzymes thought to be involved in signal transduction in vitro followed by a single cumulative concentration-response curve to the muscarinic receptor agonist carbachol. The outcome measures were the maximal contraction, the potency of carbachol, and the affinity of the M3-selective antimuscarinic agent darifenacin for inhibition of contraction. Inhibition of phosphoinositide-specific phospholipase C (PI-PLC) with 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphorylcholine (ET-18-OCH3) reduces carbachol potency and reduces darifenacin affinity, whereas inhibition of phosphatidyl choline-specific phospholipase C (PC-PLC) with O-tricyclo[5.2.1.02,6]dec-9-yl dithiocarbonate potassium salt (D609) attenuates the carbachol maximal contraction. Inhibition of rho kinase with (R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride (Y-27632) reduces carbachol potency and increases darifenacin affinity. Inhibition of rho kinase, protein kinase A (PKA), and protein kinase G (PKG) with 1-(5-isoquinolinesulfonyl)-homopiperazine·HCl (HA-1077) reduces the carbachol maximal contraction, carbachol potency, and darifenacin affinity. Inhibition of protein kinase C (PKC) with chelerythrine increases darifenacin affinity, whereas inhibition of rho kinase, PKA, PKG, and PKC with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine·2HCl (H7) reduces the carbachol maximum and carbachol potency while increasing darifenacin affinity. Inhibition of rho kinase, PKA, and PKG with N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide·2HCl (H89) reduces carbachol maximum and carbachol potency. Both the M2 and the M3 receptor subtype are involved in normal rat bladder contractions. The M3 subtype seems to mediate contraction by activation of PI-PLC, PC-PLC, and PKA, whereas the M2 signal transduction cascade may include activation of rho kinase, PKC, and an additional contractile signal transduction mechanism independent of rho kinase or PKC.

To examine the role of striatal mechanisms in cocaine-induced stereotyped licking, we investigated the acute effects of cocaine on striatal neurons in awake, freely moving rats before and after cocaine administration (0, 5, 10, or 20 mg/kg). Stereotyped licking was induced only by the high dose. Relative to control (saline), cocaine reduced lick duration and concurrently increased interlick interval, particularly at the high dose, but it did not affect licking rhythm. Firing rates of striatal neurons phasically related to licking movements were compared between matched licks before and after injection, minimizing any influence of sensorimotor variables on changes in firing. Both increases and decreases in average firing rate of striatal neurons were observed after cocaine injection, and these changes exhibited a dose-dependent pattern that strongly depended on predrug firing rate. At the middle and high doses relative to the saline group, the average firing rates of slow firing neurons were increased by cocaine, resulting from a general elevation of movement-related firing rates. In contrast, fast firing neurons showed decreased average firing rates only in the high-dose group, with reduced firing rates across the entire range for these neurons. Our findings suggest that at the high dose, increased phasic activity of slow firing striatal neurons and simultaneously reduced phasic activity of fast firing striatal neurons may contribute, respectively, to the continual initiation of stereotypic movements and the absence of longer movements.

The nicotine metabolite cotinine is an abundant long-lived bio-active compound that may contribute to the overall physiological effects of tobacco use. Although its mechanism of action in the central nervous system has not been extensively investigated, cotinine is known to evoke dopamine release in the nigrostriatal pathway through an interaction at nicotinic receptors (nAChRs). Because considerable evidence now demonstrates the presence of multiple nAChRs in the striatum, the present experiments were done to determine the subtypes through which cotinine exerts its effects in monkeys, a species that expresses similar densities of striatal α4β2* (nAChR containing the α4 and β2 subunits, but not α3 or α6) and α3/α6β2* (nAChR composed of the α3 or α6 subunits and β2) nAChRs. Competition binding studies showed that cotinine interacts with both α4β2* and α3/α6β2* nAChR subtypes in the caudate, with cotinine IC50 values for inhibition of 5-[125I]iodo-3-[2(S)-azetinylmethoxy]pyridine-2HCl ([125I]A-85380) and 125I-α-conotoxinMII binding in the micromolar range. This interaction at the receptor level is of functional significance because cotinine stimulated both α4β2* and α3/α6β2* nAChR [3H]dopamine release from caudate synaptosomes. Our results unexpectedly showed that nicotine evokes [3H]dopamine release from two α3/α6β2* nAChR populations, one of which was sensitive to cotinine and the other was not. This cotinine-insensitive subtype was only present in the medial caudate and was preferentially lost with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced nigrostriatal damage. In contrast, cotinine and nicotine elicited equivalent levels of α4β2* nAChR-mediated dopamine release. These data demonstrate that cotinine functionally discriminates between two α3/α6β2* nAChRs in monkey striatum, with the cotinine-insensitive α3/α6β2* nAChR preferentially vulnerable to nigrostriatal damage.

The effects of a specific copper(I)-chelator, neocuproine (NC), and a selective copper(II)-chelator, cuprizone, on nonadrenergic-noncholinergic transmitter mechanisms in the rat urinary bladder were studied by measuring nerve-evoked contractions of bladder strips and voiding function under urethane anesthesia. After blocking cholinergic and adrenergic transmission with atropine and guanethidine, electrical field stimulation induced bimodal contractions of bladder strips. An initial, transient contraction that was blocked by the purinergic antagonist, suramin, was significantly enhanced by NC (20 and 200 μM applied sequentially) but not affected by cuprizone. The facilitating effect, which was blocked by suramin and reversible after washout of the drug, did not occur following administration of neocuproine-copper(I) complex (NC-Cu). NC (20 μM) significantly increased the second, more sustained contraction, whereas 200 μM decreased this response. These effects of NC on the sustained contractions were not elicited by NC-Cu and not blocked by suramin. The nitric oxide synthase inhibitor, L-nitroarginine, did not alter the responses to NC. NC (20 μM) elicited a marked increase in basal tone of the strips. This effect was less prominent after the second application of 200 μM NC or with NC-Cu treatment or in the presence of suramin. In anesthetized rats, during continuous infusion cystometry, intravesical infusion of 50 μM NC but not NC-Cu or cuprizone significantly decreased the intercontraction interval (ICI) without changing contraction amplitude. The ICI returned to normal after washout of NC. Suramin blocked this effect. These results indicate that NC enhances bladder activity by facilitating purinergic excitatory responses and that copper(I)-sensitive mechanisms tonically inhibit purinergic transmission in the bladder.

Heteromerization of opioid receptors has been shown to alter opioid receptor pharmacology. However, how receptor heteromerization affects the processes of endocytosis and post-endocytic sorting has not been closely examined. This question is of particular relevance for heteromers of the mu and delta opioid receptor (MOR and DOR respectively), since the MOR is primarily recycled after endocytosis and the DOR is degraded in the lysosome. Here we examined the endocytic and post-endocytic fate of MORs, DORs and DOR/MOR heteromers in HEK293 stably expressing each receptor alone or co-expressing both receptors. We found that the clinically relevant MOR agonist methadone promotes endocytosis of MOR but also of the DOR/MOR heteromer. Furthermore, we show that DOR/MOR heteromers that are endocytosed in response to methadone are targeted for degradation, while MORs in the same cell are significantly more stable. Importantly, we found that the DOR-selective antagonist naltriben (NTB) could block both methadone- and DAMGO-induced endocytosis of the DOR/MOR heteromers but did not block signaling from this heteromer. Together, our results suggest that the MOR adopts novel trafficking properties in the context of the DOR/MOR heteromer. In addition, they suggest that the heteromer shows “biased antagonism”, whereby DOR antagonist can inhibit trafficking but not signaling of the DOR/MOR heteromer.