In vivo metabolism and clearance of substance P and co-expressed tachykinins in rat striatum.

Neurons expressing the preprotachykinin A gene, which encodes the sequences of substance P, neurokinin A, neuropeptide gamma and neuropeptide K, exemplify peptide co-existence. Furthermore, there is also evidence that substance P fragments have biological activity. However, the relative contribution of each of these peptides to tachykinin signalling is still poorly understood. An important factor which will determine the characteristics of the signal mediated by co-localised peptides is their clearance from the extracellular space. The striatum, in which tachykinins are present and exert neuromodulatory roles, can be used as a model to investigate this aspect. Therefore, in this study we characterised in vivo in the striatum the metabolism and clearance of substance P and of the other three co-expressed peptides. After intrastriatal administration of 1 pmol, tritiated substance P disappeared too rapidly for metabolites to be detected. However, when 10 nmol substance P and 1 pmol tritiated substance P were co-injected, substance P(1-4) and substance P(1-7), which are biologically active, were detected as major metabolites. Under these conditions, the rate of decay of tritiated substance P was 0.2 nmol/min. The effects of the peptidase inhibitors thiorphan, bestatin and captopril suggested that neutral endopeptidase 24.11 and aminopeptidases were involved in primary substance P cleavages, whereas angiotensin-converting enzyme was involved in secondary cleavages. The monitoring of the decay of unlabelled substance P by high-performance liquid chromatography gave a rate of 0.16 nmol/min. Using high-performance liquid chromatography with capillary electrophoresis, the rates of decay of 10 nmol neurokinin A or neuropeptide gamma were five and seven times faster than that of substance P. In contrast, over the time course of the experiment, no significant decay of neuropeptide K was detected. These results show that substance P disappears rapidly from the extracellular space, and supports the formation in vivo of major N-terminal active substance P metabolites. Our study also highlights significant differences in the clearance of co-expressed tachykinins and suggests that certain species may disappear relatively slowly from the extracellular space, and thus may make a significant temporal and spatial contribution to signalling.

N- and C-terminal substance P fragments modulate striatal dopamine outflow through a cholinergic link mediated by muscarinic receptors.

The present study investigated whether the modulatory effects of substance P and substance P fragments on striatal dopamine release involve a cholinergic link. Rat striatal slices were incubated with substance P, substance P(1-4), substance P(1-7), substance P(5-11) and substance P(8-11) in the absence or presence of various agents which modify cholinergic transmissions, and endogenous dopamine outflow was measured using high-performance liquid chromatography. The incubation of striatal slices with substance P and its N- and C-terminal fragments (1 nM) induced a significant overflow of endogenous dopamine. Neostigmine (150 nM) potentiated the effects of substance P and its fragments, whereas the incubation with hemicholinium-3 (50 microM) abolished the effects of the peptides on dopamine outflow. The acetylcholinesterase inhibitor and the inhibitor of choline uptake did not have intrinsic effects on dopamine outflow. The muscarinic antagonist atropine (1 microM) reversed completely the effects of substance P and its fragments, whereas the nicotinic antagonists dihydro-beta-erythroidine (0.5 microM) and pempidine (10 microM) were devoid of effects. None of the cholinergic antagonists modified dopamine outflow. The results suggest that substance P and several N- and C-terminal substance P fragments activate cholinergic neurons in striatal slices. The released acetylcholine induces an increased dopamine outflow, mediated by muscarinic receptors. These observations represent additional evidence which supports the functional interactions between substance P, acetylcholine and dopamine in the striatum. Furthermore, they show that substance P fragments may exert neuromodulatory effects through mechanisms similar to those underlying the effects of the parent peptide.

Imipramine and phenelzine decrease glutamate overflow in the prefrontal cortex--a possible mechanism of neuroprotection in major depression?

Antidepressant drugs have been used for decades, but the neurobiological substrate of their efficacy is not completely understood. Although these drugs have well-established effects on monoamines, evidence is emerging that they may also affect other neurotransmitter systems. It has been shown that treatment with a wide range of antidepressants changes the binding characteristics of the N-methyl-D-aspartate type of glutamate receptor. This change is delayed and occurs only in the cortex. The mechanism that triggers it is unknown. We hypothesized that N-methyl-D-aspartate receptor alterations may be due to changes in the dynamics of cortical excitatory amino acid release. Such changes are of particular interest in areas such as the prefrontal cortex, a region involved in stress responses and affected in major depression. We investigated the effects of two antidepressants with different modes of action, imipramine and phenelzine, on glutamate and aspartate outflow in rat prefrontal cortex and striatum. We showed that antidepressants significantly decreased stimulated glutamate outflow. The effect had a rapid onset, was sustained during chronic administration and was only seen in the prefrontal cortex. This change may initiate receptor alterations. Furthermore, if antidepressants can dampen states of hyperglutamatergic activity and the subsequent excitotoxicity, their chronic use may have a considerable neuroprotective potential in major depression.

Behavioural and pharmacokinetic studies in the monkey (Macaca mulatta) with diazepam, nordiazepam and related 1,4-benzodiazepines.

1. Behavioural activity (delayed differentiation and spatial delayed alternation) and pharmacokinetics of diazepam and its metabolites, N-desmethyldiazepam (nordiazepam), 3-hydroxydiazepam (temazepam) and 3-hydroxy-N-desmethyldiazepam (oxazepam), and of dipotassium clorazepate (clorazepate), were studied in the monkey (Macaca mulatta). Diazepam and its metabolites (1.8 and 3.0 mg/kg) and clorazepate (2.6 and 4.3 mg/kg) were given by intraperitoneal injection. 2. Hydroxylation of diazepam (temazepam and oxazepam) led to a loss of, or a considerable reduction in, behavioural activity, whereas activity was preserved, though modified, by demethylation (nordiazepam). It was not possible to establish change in behaviour at specific time intervals after clorazepate, but combined performance data revealed an effect. 3. The maximum mean plasma concentrations of diazepam, temazepam, oxazepam and clorazepate were observed at 0.5 h, and the maximum mean plasma concentration of nordiazepam was observed at 1 hour. Plasma concentrations of nordiazepam were the highest and decreased monoexponentially. Plasma concenqrations of the other drugs declined rapidly at first but more slowly later, and these data were analysed as biexponential models. In the analysis for metabolites, nordiazepam reached measurable levels after the injection of diazepam and clorazepate. 4. It is suggested that differences in the effects of closely related benzodiazepines may not be due solely to their plasma pharmacokinetic properties, but may arise from differences in their intrinsic activity.

Biphasic inhibition of stimulated endogenous dopamine release by 7-OH-DPAT in slices of rat nucleus accumbens.

1. Fast cyclic voltammetry was used to investigate the effect of 7-OH-DPAT (7-hydroxy-N,N-di-n-propyl-2-aminotetralin), a putative D3 receptor agonist, on electrically stimulated endogenous dopamine release in slices of rat nucleus accumbens. 2. 7-OH-DPAT inhibited single pulse stimulated dopamine release in a concentration-dependent manner with a maximum inhibition of 95.5%. Analysis of concentration-response curves to 7-OH-DPAT showed that they were biphasic, with the high affinity component contributing 18.0% to the total inhibition and the low affinity component 77.5%. 7-OH-DPAT exhibited a 560 fold selectivity between the high and low affinity components (0.015 nM compared to 8.4 nM). 3. Concentration-response curves to the non-selective D2/D3 agonist, apomorphine, were monophasic. The maximum inhibition was 93.1% and the EC50 value 82 nM. 4. The selective D2 antagonist, haloperidol (30 nM), antagonized the low affinity component of the concentration-response cuve to 7-OH-DPAT whilst the high affinity component was essentially unaffected. The pKB values calculated for the high and low affinity components were 7.89 and 9.45 respectively. 5. In conclusion, these results demonstrate that 7-OH-DPAT inhibits stimulated dopamine release by acting at two different sites. Furthermore, the results are consistent with the hypothesis that the high and low affinity components of the concentration-response curve to 7-OH-DPAT may reflect activation of functional D3 and D2 release-regulating autoreceptors respectively. However, the possibility that the biphasic nature of the curve may reflect different subtypes of the D2 receptor cannot be excluded.

Effects of chlorpromazine and promazine on the visual aftereffects of tilt and movement.

The effects of chlorpromazine (CPZ) and promazine on the visual aftereffects of tilt and motion were measured. CPZ markedly reduced the strength of both aftereffects, while promazine produced a smaller and not always significant reduction. Control experiments suggested that the effects were produced in the central visual system rather than by several possible peripheral artefacts or by drowsiness. The effects are discussed with reference to the pharmacological activity of the drugs and their influence on the strength of inhibition in the visual cortex, both in normal subjects and in schizophrenic illness.

A model of gastric emptying using paracetamol absorption in intensive care patients.

OBJECTIVES: To describe the range and factors which may affect gastric emptying in the ICU patient. DESIGN: Validation sample. SETTING: The adult Intensive Care Unit (ICU) of a teaching hospital. PATIENTS: Twenty-seven ICU patients, aged 18-65 years were studied within 3 days of their ICU admission. All patients had normal hepatic and renal chemistry and had no contraindications to enteral feeding. MEASUREMENTS AND MAIN RESULTS: The area under the concentration curve from 0-60 min (AUC60) of a paracetamol absorption test was used as the measure of gastric emptying. The variables of the presence or absence of bowel sounds, volume of gastric aspirate ( > 50 ml or < 50 ml), an estimated risk of death (ROD), an APACHE II score calculated 24 h before the study, a pHi measurement, the use of dopamine (2.5-5 microg/kg, yes or no) and of opioids were included in a multiple regression analysis. Using Pearson correlation, AUC60 was positively correlated with the estimated ROD (r = 0.50, p < 0.05). There was a statistically significant difference in the mean AUC60 between those patients who did, and those who did not, receive dopamine (t = 3.06, p < 0.005). On multiple regression analysis the only variable which was significantly associated with AUC60 was estimated ROD, which accounted for 25% of the variance in AUC60. CONCLUSION: The results suggest that there is a wide range in gastric emptying in critically ill patients. The results may be due to the case mix of the patients. The use of dopamine may adversely affect gastric emptying and requires further investigation in the ICU patient. Prediction of gastric emptying is difficult in these patients and further investigation is necessary in order to improve our understanding of this process.

N- and C-terminal substance P fragments: differential effects on striatal [3H]substance P binding and NK1 receptor internalization.

N- and C-terminal substance P (SP) fragments increase striatal dopamine outflow at nanomolar concentrations. This contrasts with their low affinity for NK1 receptors. To explore this discrepancy, we investigated the interaction of SP and SP fragments with NK1 sites in fresh striatal slices, the same model used in the functional studies on dopamine outflow. [3H]SP bound specifically to one site (Kd = 6.6 +/- 0.9 nM; Bmax = 12.6 +/- 0.7 fmol/mg protein). [3H]SP binding was displaced by SP (IC50 = 11.8 nM), but not by SP(1-7) or SP(5-11), up to 10 microM. In contrast, 10 nM SP(1-7) or SP(5-11) induced significant internalization of the NK1 receptor, similar to that induced by SP. We suggest that SP fragments have high affinity for an NK1 receptor conformer which is different from that labelled by [3H]SP.

Substance P fragments and striatal endogenous dopamine outflow: interaction with substance P.

Accumulating evidence shows that N- and C-terminal substance P fragments have significant biological activity. Substance P(1-9) and substance P(6-11) have been reported to be major substance P metabolites in rat striatum. We investigated the effects of these fragments on endogenous dopamine outflow in rat striatal slices. Substance P-(1-9) and substance P-(6-11) induced a significant increase in dopamine outflow at 0.1 and 1 nM. The effects of substance P-(6-11) (1 nM) were reversed by the tachykinin NK1 antagonist WIN 51,708 (17beta-hydroxy-17alpha-ethynyl-5alpha-androstano[3,2- b]pyrimido[1,2-a]benzimidazole) (2.5 nM), whereas the effects of substance P-(1-9) were not modified by the antagonist. Substance P-(1-9) and substance P-(6-11) (1 nM) did not increase the dopamine overflow induced by 25 mM KCI. The effects of the two fragments were reversed by the muscarinic antagonist atropine (1 microM) but not by nicotinic antagonists dihydro-beta-erythroidine (0.5 microM) and pempidine (10 microM). The co-incubation of tissue with substance P and each fragment in a 1/1 or 10/1 ratio of substance P to metabolite revealed a negative interaction between parent and fragments. A similar pattern was observed when substance P was co-administered with the active fragments substance P(1-4), substance P(1-7), substance P(5-11) and substance P(8-11). The data show that substance P-(1-9) and substance P-(6-11) have modulatory effects similar to substance P. However, the presence of active substance P metabolites does not appear to amplify the signal mediated by the parent peptide.

Substance P-(1-7) and substance P-(5-11) locally modulate dopamine release in rat striatum.

The effects of substance P, substance P-(1-7) and substance P-(5-11) on endogenous dopamine outflow in rat striatal slices were investigated. The dose-response curves (0.01 nM to 10 microM) were bell-shaped, with significant increases at 0.1 and 1 nM but with no effect at higher concentrations. The tachykinin NK1 receptor agonist, [Sar9,Met(O2)11]substance P, significantly increased dopamine outflow at 10 and 100 nM. The effects of substance P or substance P-(5-11) and 25 mM KCl were additive. A negative interaction was observed with substance P-(1-7) and K+. The increase in dopamine outflow elicited by 1 nM substance P and substance P-(5-11) was reversed by the tachykinin NK1 receptor antagonist WIN 51,708 (17 beta-hydroxy-17 alpha-ethynyl-5 alpha-androstano[3,2-b]pyrimido[1,2- alpha]benzimidazole) (25 and 250 nM), whereas only partial reversal was observed for the effect of substance P-(1-7). These results show that substance P fragments locally modulate striatal dopamine outflow and the mechanisms underlying this modulation may differ between N- and C-terminal fragments.

Evidence for modulatory effects of substance P fragments (1-4) and (8-11) on endogenous dopamine outflow in rat striatal slices.

The effects of substance P-(1-4) and substance P-(8-11) on endogenous dopamine outflow in rat striatal slices were investigated. The dose-response curves (0.01 nM to 1 microM) were bell-shaped for both peptides, with significant increases in dopamine outflow at 0.1 and 1 nM. Dopamine overflow elicited by 1 nM substance P-(1-4) or substance P-(8-11) and 25 mM KCl was additive. Although substance P-(8-11) contains a truncated tachykinin sequence, the tachykinin NK1 receptor antagonist WIN 51,708 (17 beta-hydroxy-17 alpha-ethynyl-5 alpha-androstano[3,2-b]pyrimido[1,2- a]benzimidazole (2.5 nM) fully reversed its effect. The interaction between the antagonist and 1 nM substance P-(1-4) was statistically not significant. The data constitute the first evidence that the fragments substance P-(1-4) and substance P-(8-11) could exert central effects and suggest that they may play a role in neuromodulation in the basal ganglia.

Substance P modulation of striatal dopamine outflow is determined by M(1) and M(2) muscarinic receptors in male wistar rats.

Substance P (SP) stimulates striatal dopamine outflow through a cholinergic muscarinic link. SP-induced increase in acetylcholine (Ach) is concentration-dependent, whereas the stimulation of dopamine outflow is seen only over a limited concentration range. M(1) and M(2) receptor stimulation has opposite effects on dopamine outflow. We postulated that the effect of SP on dopamine outflow depends on the M(1)/M(2) balance. We show that Ach (10-2500 microM) stimulates dopamine outflow in striatal slices in a biphasic manner, similar to SP (0.01-100 nM). An inactive SP concentration (10 nM) which was higher than the active concentration range, became active in the presence of the M(2) antagonist methoctramine (100 microM). Conversely, the effect of 1 nM SP was reversed by the M(1) antagonist pirenzepine (1 microM). Our observations show that SP modulation of dopamine outflow is determined by a balance between M(1) and M(2) receptors.

Distribution of temoporfin, a new photosensitizer for the photodynamic therapy of cancer, in a murine tumor model.

The biodistribution of temoporfin (tetra[m-hydroxyphenyl]chlorin, m-THPC), a recently developed photosensitizer, was investigated in BALB/c mice. The drug was administered intravenously (0.35-0.75 mumol/kg) to tumor-free mice or to mice implanted with the Colo 26 colorectal carcinoma. Blood and tissue samples were collected for up to 96 h post-dose. Drug concentrations were determined by HPLC coupled to photometric detection at 423 nm. Concentrations in blood and liver fell relatively rapidly such that blood concentrations at later time points were below the limit of detection. Tumor concentrations rose at first and then remained constant from 24 h. Temoporfin concentrations in some tissues, notably heart and skeletal muscle, declined only slowly when compared to blood. The tumor: tissue ratios for those organs that showed a more rapid decline in temoporfin concentrations were higher at later times, whereas in tissues such as muscle the ratio remained relatively constant. The organs with the highest tumor:tissue ratios were small intestine (8.6), liver (6.9) and skeletal muscle (5.0).

Distribution and excretion of radiolabeled temoporfin in a murine tumor model.

The biodistribution and excretion of temoporfin (tetra[m-hydroxyphenyl]chlorin, m-THPC), a recently developed photosensitizer, was investigated in BALB/c mice. [14C]temoporfin was administered intravenously (0.73 mumol/kg) to tumor-free mice or to mice implanted with the Colo 26 colorectal carcinoma. Blood, tissue and fecal samples were collected for 35 days and 10 days postdose from tumor-free mice and tumor-bearing mice, respectively. Blood concentrations fell rapidly such that at later time points they were indistinguishable from background counts. Tumor concentrations rose to a peak of 0.34 microgram temoporfin equivalents/mL at 2 days and then declined in parallel (log plot) with the blood concentrations. Tumor: tissue ratios at 2 days for skin, adipose tissue and skeletal muscle underlying the tumor were 1.5, 2.3 and 3.8, respectively. By 4 days the corresponding values were 1.6, 3.4 and 4.0. Nearly 40% of the administered radioactivity was excreted in the feces in the first 24 h and more than 80% had been excreted by 20 days. Less than 0.2% of the dose was recovered from the urine. An elimination half-life of 10-12 days was calculated from the excretion data.

Analysis of solutions containing glutathione and inorganic nitrite: application to nitroglycerin metabolism studies.

Nitroglycerin (GTN) is metabolized to 1,2-dinitroglycerin (1,2-GDN) and 1,3-dinitroglycerin (1,3-GDN) in vivo and in liver homogenates. 1,2-GDN and 1,3-GDN are converted to isomers of glyceryl mononitrate (GMN) in vivo. The denitration reactions yield inorganic nitrite (NO(-)(2)) which is oxidized to inorganic nitrate (NO(-)(3)). Denitration involves utilization of glutathione (GSH). In attempting to use the Bratton-Marshall assay for NO(-)(2) in studies of GTN metabolism in vitro, and in attempting to use Ellman's reagent for GSH in the same research, apparent concentrations of both NO(-)(2) and GSH were noticed lower than anticipated. Apparent mutual interference by NO(-)(2) and GSH in their respective assays was then found. Development of a specific liquid chromatographic method for measurement of NO(-)(2), NO(-)(3), GSH and oxidized glutathione (GSSG) permitted the study of the interaction of NO(-)(2) and GSH, which yielded NO(-)(3) and GSSG.

Ionization constants, octanol partition coefficients and cholinesterase inhibitor constants for chlorpromazine and its metabolites.

The relationship between the lipophilic character of chlorpromazine and seven of its metabolites and their ability to inhibit horse serum cholinesterase (Ki) has been investigated. Log(1/Ki) values were correlated with log P octanol partition coefficients (r = 0.88, P less than 0.01, n = 8). The inhibitor values ranged from 2.7 x 10(-6) M for chlorpromazine to 48.6 x 10(-6) M for monodesmethylchlorpromazine sulphoxide. Ionization constants were determined by limiting solubility, spectrophotometry and pH-partition characteristics. Demethylated metabolites were more basic than the tertiary amines and the sulphoxides were slightly less basic than the corresponding sulphides.

The binding of physostigmine to human serum albumin.

The binding of [3H]physostigmine to crystallized human serum albumin (HSA) has been investigated using equilibrium dialysis. The percentage bound to 1% (w/v) HSA decreased from 18 to 4% as the total concentration of physostigmine increased from 3.3 nM to 2.7 microM (0.9 to 750 ng mL-1). A single class of specific binding sites with a large affinity constant, K = 8 x 10(7) L mol-1, was identified. The concentration of binding sites was approximately 3 nM. The Michaelis constants for human serum cholinesterase and albumin were the same; an explanation for these results is that the drug is binding to a trace cholinesterase, in the albumin.

Methods for study of fluphenazine kinetics in man.

Fluphenazine and its principal metabolites, fluphenazine sulphoxide, and 7-hydroxyfluphenazine were identified and quantified in human plasma, urine and faeces following intramuscular and oral administration of 14C-fluphenazine dihydrochloride. The presence of a conjugate fraction was also noted. Unmetabolized fluphenazine was selectively extracted into n-heptane. The metabolites were separated by solvent extraction into toluene. Conjugates were hydrolysed back to fluphenazine, fluphenazne sulphoxide and 7-hydroxyfluphenazine. Fluphenazine and fluphenazine conjugates were also measured in the urine of patients receiving long term non-radioactive fluphenazine decanoate therapy. The urinary excretion rate of the conjugate fraction was systematically related to the plasma concentration, regardless of urine flow rate or pH, providing a convenient method for the assessment of fluphenazine kinetics by urinary excretion studies not involving administration of labelled drug.

Chromatographic separation of thioridazine sulphoxide and N-oxide diastereoisomers: identification as metabolites in the rat.

(+/-)-Thioridazine and (+/-)-desmethylthioridazine have been oxidized to produce a number of chiral sulphoxide and amine oxide compounds. Diastereoisomeric isomers were separated by thin-layer and high performance liquid chromatography. Thioridazine-5-sulphoxide, N-desmethylthioridazine-5-sulphoxide and thioridazine-N-oxide diastereoisomers were found to be thioridazine metabolites following dosing in rats or after in-vitro incubation with rat liver homogenate.

Are intraligamentary injections intravascular?

A pressure type syringe was used to give intraligamentary injections (IL) to upper teeth of two formulations commonly used in general practice, lignocaine and prilocaine. Assay of plasma levels of drug was carried out by high performance liquid chromatography. Results of assays after intraligamentary injections were then compared with results of assays after intravenous injections of plain drug in the same subjects. Both formulations of local anaesthetic were found as peak levels in the circulation, presumably after intraosseous spread, by 2 minutes following the intraligamentary injections. For lignocaine the peak amount was nearly 7% of the intravenous dose and for prilocaine the peak amount was 25% of the intravenous dose, at 2 minutes after injection. It was concluded that IL injections for healthy adults were unlikely to cause systemic unwanted effects when given in small doses.