Relativistic quantum revivals.

Quantum revivals are now a well-known phenomena within nonrelativistic quantum theory. In this Letter we display the effects of relativity on revivals and quantum carpets. It is generally believed that revivals do not occur within a relativistic regime. Here we show that while this is generally true, it is possible, in principle, to set up wave packets with specific mathematical properties that do exhibit exact revivals within a fully relativistic theory.

Agonist binding, agonist affinity and agonist efficacy at G protein-coupled receptors.

Measurements of affinity and efficacy are fundamental for work on agonists both in drug discovery and in basic studies on receptors. In this review I wish to consider methods for measuring affinity and efficacy at G protein coupled receptors (GPCRs). Agonist affinity may be estimated in terms of the dissociation constant for agonist binding to a receptor using ligand binding or functional assays. It has, however, been suggested that measurements of affinity are always contaminated by efficacy so that it is impossible to separate the two parameters. Here I show that for many GPCRs, if receptor/G protein coupling is suppressed, experimental measurements of agonist affinity using ligand binding (K(obs)) provide quite accurate measures of the agonist microscopic dissociation constant (KA). Also in pharmacological functional studies, good estimates of agonist dissociation constants are possible. Efficacy can be quantitated in several ways based on functional data (maximal effect of the agonist (E(max)), ratio of agonist dissociation constant to concentration of agonist giving half maximal effect in functional assay (K(obs)/EC50), a combined parameter E(max)K(obs)/EC50). Here I show that E(max)K(obs)/EC50 provides the best assessment of efficacy for a range of agonists across the full range of efficacy for full to partial agonists. Considerable evidence now suggests that ligand efficacy may be dependent on the pathway used to assess it. The efficacy of a ligand may, therefore, be multidimensional. It is still, however, necessary to have accurate measures of efficacy in different pathways.

Mechanisms of G protein activation via the D2 dopamine receptor: evidence for persistent receptor/G protein interaction after agonist stimulation.

BACKGROUND AND PURPOSE: The aim of this report is to study mechanisms of G protein activation by agonists. EXPERIMENTAL APPROACH: The association and dissociation of guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding at G proteins in membranes of CHO cells stably transfected with the human dopamine D(2short) receptor was studied in the presence of a range of agonists. KEY RESULTS: Binding of [(35)S]GTPgammaS was dissociable in the absence of agonist and dissociation was accelerated both in rate and extent by dopamine, an effect which was blocked by the dopamine D(2) receptor antagonist raclopride and by suramin, which inhibits receptor/G protein interaction. A range of agonists of varying efficacy increased the rate of dissociation of [(35)S]GTPgammaS binding, with the more efficacious agonists resulting in faster dissociation. Agonists were able to dissociate about 70% of the pre-bound [(35)S]GTPgammaS, leaving a component which may not be accessible to the agonist-bound receptor. The dissociable component of the [(35)S]GTPgammaS binding was reduced with longer association times and increased [(35)S]GTPgammaS concentrations. CONCLUSIONS AND IMPLICATIONS: These data are consistent with [(35)S]GTPgammaS binding being initially to receptor-linked G proteins and then to G proteins which have separated from the agonist bound receptor. Under the conditions used typically for [(35)S]GTPgammaS binding assays, therefore, much of the agonist-receptor complex remains in proximity to G proteins after they have been activated by agonist.

Analysis of second messenger pathways stimulated by different chemokines acting at the chemokine receptor CCR5.

The chemokine receptor, CCR5, responds to several chemokines leading to changes in activity in several signalling pathways. Here, we investigated the ability of different chemokines to provide differential activation of pathways. The effects of five CC chemokines acting at CCR5 were investigated for their ability to inhibit forskolin-stimulated 3'-5'-cyclic adenosine monophosphate (cAMP) accumulation and to stimulate Ca(2+) mobilisation in Chinese hamster ovary (CHO) cells expressing CCR5. Macrophage inflammatory protein 1alpha (D26A) (MIP-1alpha (D26A), CCL3 (D26A)), regulated on activation, normal T-cell expressed and secreted (RANTES, CCL5), MIP-1beta (CCL4) and monocyte chemoattractant protein 2 (MCP-2, CCL8) were able to inhibit forskolin-stimulated cAMP accumulation, whilst MCP-4 (CCL13) could not elicit a response. CCL3 (D26A), CCL4, CCL5, CCL8 and CCL13 were able to stimulate Ca(2+) mobilisation through CCR5, although CCL3 (D26A) and CCL5 exhibited biphasic concentration-response curves. The Ca(2+) responses induced by CCL4, CCL5, CCL8 and CCL13 were abolished by pertussis toxin, whereas the response to CCL3 (D26A) was only partially inhibited by pertussis toxin, indicating G(i/o)-independent signalling induced by this chemokine. Although the rank order of potency of chemokines was similar between the two assays, certain chemokines displayed different pharmacological profiles in cAMP inhibition and Ca(2+) mobilisation assays. For instance, whilst CCL13 could not inhibit forskolin-stimulated cAMP accumulation, this chemokine was able to induce Ca(2+) mobilisation via CCR5. It is concluded that different chemokines acting at CCR5 can induce different pharmacological responses, which may account for the broad spectrum of chemokines that can act at CCR5.

Aspects of the structure of the D2 dopamine receptor.

Significant new information on the D2 dopamine receptor has recently become available from a combination of protein chemical and molecular genetic analyses. Molecular genetic studies have shown the receptor to be a member of the family of receptors that are linked to G proteins and that have structures predicted to contain seven transmembrane domains. Two distinct species of D2 dopamine receptor have been found which may differ in their coupling to G proteins; their distributions have been mapped at the nucleic acid level. The D2 dopamine receptor has been purified from brain and anterior pituitary and characterized. Chemical modification of the brain receptor provides evidence for the importance of a carboxyl group that interacts with ligands at the receptor binding site. Here, Philip Strange discusses these points and proposes models of receptor-ligand interaction based on the conservation of several aspartic acid residues in receptors that bind cationic amines.

Assays for enhanced activity of low efficacy partial agonists at the D(2) dopamine receptor.

BACKGROUND AND PURPOSE: Low efficacy partial agonists at the D2 dopamine receptor may be useful for treating schizophrenia. In this report we describe a method for assessing the efficacy of these compounds based on stimulation of [35S]GTPgammaS binding. EXPERIMENTAL APPROACH: Agonist efficacy was assessed from [(35)S]GTPgammaS binding to membranes of CHO cells expressing D2 dopamine receptors in buffers with and without Na+. Effects of Na+ on receptor/G protein coupling were assessed using agonist/[3H]spiperone competition binding assays. KEY RESULTS: When [35S]GTPgammaS binding assays were performed in buffers containing Na+, some agonists (aripiprazole, AJ-76, UH-232) exhibited very low efficacy whereas other agonists exhibited measurable efficacy. When Na+ was substituted by N-methyl D-glucamine, the efficacy of all agonists increased (relative to that of dopamine) but particularly for aripiprazole, aplindore, AJ-76, (-)-3-PPP and UH-232. In ligand binding assays, substitution of Na+ by N-methyl D-glucamine increased receptor/G protein coupling for some agonists -. aplindore, dopamine and (-)-3-PPP - but for aripiprazole, AJ-76 and UH-232 there was little effect on receptor/G protein coupling. CONCLUSIONS AND IMPLICATIONS: Substitution of Na+ by NMDG increases sensitivity in [(35)S]GTPgammaS binding assays so that very low efficacy agonists were detected clearly. For some agonists the effect seems to be mediated via enhanced receptor/G protein coupling whereas for others the effect is mediated at another point in the G protein activation cycle. AJ-76, aripiprazole and UH-232 seem particularly sensitive to this change in assay conditions. This work provides a new method to discover these very low efficacy agonists.

Reduction in accumulation of [3H]triphenylmethylphosphonium cation in neuroblastoma cells caused by optical probes of membrane potential. Evidence for interactions between carbocyanine dyes and lipophilic anions.

The accumulation of [3H]triphenylmethylphosphonium cation in neuroblastoma N1E 115 cells in the presence of tetraphenylboron is reduced by 3,3'-diethylthiadicarbocyanine iodide and by 3,3'-dipropylthiadicarbocyanine iodide. This reduction in uptake of the lipophilic cation is not due to the carbocyanine dyes depolarizing the plasma membrane of these cells but due to an interaction between the carbocyanine dyes and tetraphenylboron leaving less of the lipophilic anion free in solution to assist uptake of the lipophilic cation. This interaction is shown to have a 1:1 stoicheiometry.

The energetics of ligand binding at catecholamine receptors.

One of the key events in the actions of agonists and antagonists is their binding to receptors. Understanding this event is of interest in terms of understanding receptor function but it also has immense practical relevance for the design of drugs. If the ligand-binding process could be understood in detail, including the nature of the interactions made between ligand and receptor, then this could help in the design of more-selective drugs. The interaction of a ligand with its receptor is clearly of importance in determining the specificity of ligand action but ligand-receptor interaction also initiates the processes of signalling that are exhibited in the efficacy of ligand action. Here Philip Strange considers these events for catecholamine receptors, concentrating mostly on dopamine receptors; where necessary the discussion is widened to include other receptor systems.

Pathology and drug action in schizophrenia: insights from molecular biology.

Schizophrenia is a severe disorder of personality which has a genetic basis. Schizophrenia arises from a change in brain development. There is no strong evidence that disturbances in neurotransmitter systems are a primary cause. Anti-psychotic drugs act primarily through D2 and D3 dopamine receptors. The atypical drug clozapine may act through a number of different receptors, including D2, D3 and D4 dopamine receptors. Anti-psychotic drugs are inverse agonists at D2 dopamine receptors.

Receptors for neurotransmitters and related substances.

Advances in techniques for cloning neurotransmitter receptors have revealed new targets for selective drug design. Cell systems for more efficient expression of cloned receptor genes have also been developed. Knowledge of the nature of ligand-binding sites is now becoming available and this should aid in the design of better drugs with fewer side effects.

Efficacy, safety, and pump compatibility of insulin aspart used in continuous subcutaneous insulin infusion therapy in patients with type 1 diabetes.

OBJECTIVE: The purpose of this study was to compare the efficacy, safety and pump compatibility of insulin aspart (a rapid-acting insulin analog) and buffered regular human insulin in patients with type 1 diabetes undergoing continuous subcutaneous insulin infusion (CSII) therapy. RESEARCH DESIGN AND METHODS: This was a single-center randomized open-label study Patients received CSII therapy with insulin aspart (n = 19) or buffered regular human insulin (n = 10) for 7 weeks. Bolus doses of insulin aspart were administered immediately before meals and buffered regular human insulin 30 min before meals. RESULTS: Insulin aspart and buffered regular human insulin were both effective in controlling average daily blood glucose levels (8.2 +/- 1.9 and 8.5 +/- 2.1 mmol/l, respectively) (mean +/- SD) and maintaining serum fructosamine (343 +/- 25.7 and 336 +/- 27.4 micromol/l) and HbA1c (6.9 +/- 0.6 and 7.1 +/- 0.6%) levels. Possible obstructions and set leakages were infrequently reported in both groups. Similar numbers of patients experienced hypoglycemia (blood glucose <2.5 mmol/l): 14 (74%) insulin aspart patients versus 6 (60%) buffered regular human insulin patients. Patients receiving insulin aspart had fewer hypoglycemic events per patient (2.9) than those patients receiving buffered regular human insulin (6.2). There were no differences between the two insulins in the occurrence of hyperglycemic events (blood glucose >19 mmol/l) or in the number and type of adverse events. CONCLUSIONS: Insulin aspart and buffered regular human insulin were effective and well tolerated and provided similar pump compatibility when used in CSII therapy.

A randomized placebo-controlled trial of repaglinide in the treatment of type 2 diabetes.

OBJECTIVE: The objective of the study was to assess the efficacy and safety of repaglinide compared with placebo in the treatment of patients with type 2 diabetes. RESEARCH DESIGN AND METHODS: This was a phase II multicenter, double-blind, placebo-controlled, randomized, dose-adjustment and maintenance trial. After screening and a 2-week washout period, 99 patients were randomized to receive either repaglinide (n = 66) or placebo (n = 33). Patients underwent 6 weeks of dose adjustment followed by 12 weeks of dose maintenance. Fasting and stimulated glycosylated hemoglobin (HbA1c), plasma glucose, insulin, and C-peptide were measured at predetermined intervals. Adverse events and hypoglycemic episodes were recorded. RESULTS: From baseline to last visit, mean HbA1c decreased from 8.5 to 7.8% in patients treated with repaglinide and increased from 8.1 to 9.3% in patients receiving placebo, with a statistically significant difference of - 1.7% (P < 0.0001) between treatment groups at the last visit. Mean fasting plasma glucose and postprandial glucose increased in patients receiving placebo and decreased in patients treated with repaglinide, with statistically significant (P < 0.01) differences between groups at the last visit. Concentrations of fasting and postprandial insulin and C-peptide were lower at the last visit compared with baseline for patients treated with placebo and higher for patients treated with repaglinide, and the differences between groups were statistically significant (P < 0.05). Overall, repaglinide was well tolerated. CONCLUSIONS: This study demonstrated that repaglinide was safe and efficacious in lowering blood glucose concentrations. In addition to overall improvement in glycemic control noted with repaglinide in both sulfonylurea-treated patients and oral hypoglycemic agent-naive patients, repaglinide had a potent glucose-lowering effect in the postprandial period.

Insulin aspart (B28 asp-insulin): a fast-acting analog of human insulin: absorption kinetics and action profile compared with regular human insulin in healthy nondiabetic subjects.

OBJECTIVE: To study the pharmacokinetic and pharmacodynamic profile of insulin aspart (a new fast-acting human insulin analog) after subcutaneous administration in the deltoid, abdominal, and thigh sites and to compare this profile with regular human insulin (Novolin; Novo Nordisk A/S, Copenhagen). RESEARCH DESIGN AND METHODS: A total of 20 healthy subjects were studied in a single-center six-period double-blind randomized crossover trial with 6 study days and a washout period of 1 week between each single daily dose of the trial drug. Subjects were randomized to receive a single dose of 0.2 U/kg of insulin aspart or regular insulin on each of the 6 study days in three different sites (the deltoid, the abdomen, and the thigh) during a 10-h euglycemic clamp (two drugs and three injection sites). Pharmacokinetic and pharmacodynamic measurements were derived from blood sample measurements of glucose, insulin, and C-peptide during these clamps. RESULTS: The pharmacodynamic data from the euglycemic clamp study showed that, regardless of injection site, the maximal glucose infusion rate (GIR Cmax) was greater and occurred at an earlier time (GIR Tmax) after administration of insulin aspart than regular insulin (GIR Cmax: abdomen 813 vs. 708, deltoid 861 vs. 736, and thigh 857 vs. 720 g/min, P < 0.05 for all; GIR Tmax: abdomen 94 vs. 173, deltoid 111 vs. 192, and thigh 145 vs. 193 g/min, P < 0.05 for all). Pharmacokinetic parameters were also consistent with faster absorption and higher peak insulin concentrations after insulin aspart administration. From all sites, the peak insulin concentration (Cmax) was higher and occurred earlier (Tmax) after administration of insulin aspart than of regular insulin (Cmax: abdomen 501 vs. 260, deltoid 506 vs. 252, thigh 422 vs. 220 pmol/l, P < 0.001 for all sites; Tmax: abdomen 52 vs. 109, deltoid 54 vs. 98, and thigh 60 vs. 107 min, P < 0.01 for all sites). The absorption and glucose-lowering action of insulin aspart did not differ between sites (similar GIR Cmax, Tmax, and area under the curve parameters). However, the duration of the glucose-lowering effect was up to 34 min shorter (P < 0.01) for the abdomen injections than for the deltoid or thigh injections (lower time of 50% glucose disposal). In addition, the amount of glucose infused was significantly lower by 10-14% in the abdomen than in other sites. CONCLUSIONS: Subcutaneous administration of insulin aspart causes a more rapid and intense maximal effect compared with regular insulin during euglycemic clamp studies in nondiabetic subjects. Abdominal administration of insulin aspart has a shorter duration of glucose-lowering effect compared with administration in the deltoid or thigh.

Interferon gamma-treated keratinocytes activate T cells in the presence of superantigens: involvement of major histocompatibility complex class II molecules.

During inflammation in the skin keratinocytes can express major histocompatibility complex class II molecules but are unable to present nominal antigens to resting T cells. Certain bacteria including staphylococci produce a new class of antigens termed superantigens that are very potent T-cell activators. Using an in vitro model with cultured normal human keratinocytes and purified allogeneic T cells, we demonstrated that major histocompatibility complex class II+ keratinocytes can activate T cells in the presence of the superantigen staphylococcal enterotoxin B. Major histocompatibility complex class II+ keratinocytes activated T cells at concentrations of staphylococcal enterotoxin B as low as 100 pg/ml. The activation required contact between keratinocytes and T cells, was inhibited with a monoclonal antibody to human leukocyte antigen DR, -DQ, and was not affected by fixation of the keratinocytes. These data show that major histocompatibility complex class II+ keratinocytes activate T cells in the presence of the superantigen staphylococcal enterotoxin B.

T-lymphocyte clones initiated from lesional psoriatic skin release growth factors that induce keratinocyte proliferation.

To investigate whether growth factors derived from T cells in psoriatic lesions are able to stimulate keratinocyte growth, T-cell lines were initiated from lesional psoriasis skin and cloned by limiting dilution. Eight clones with good proliferative capacity out of 40 clones from one patient were stimulated. After 24 h, the conditioned medium was harvested and the growth modulatory effect of the conditioned medium on keratinocytes was assessed. Seven of the eight T-cell clones stimulated keratinocyte growth to an extent ranging from 22% +/- 19 to 64% +/- 9 (mean +/- SD of three experiments) of maximal inducible keratinocyte growth, and one T-cell clone had no effect (-5% +/- 2) on keratinocyte growth. Keratinocyte growth was also induced by T-cell clones obtained from two other patients. Several cytokines were tested in this system to determine which T-cell growth factor may induce the keratinocyte growth. None of the cytokines interferon-g, transforming growth factor-beta, interleukin (IL)-2, IL-3, IL-4, IL-6, IL-8, or granulocyte-macrophage colony stimulating factor alone was found to possibly be responsible for the T-cell-induced keratinocyte growth. Thus the nature of the T-cell keratinocyte growth-promoting stimulus remains to be elucidated.

Pharmacokinetic profiles of repaglinide in elderly subjects with type 2 diabetes.

Pharmacokinetic profiles of single- and multiple-dose regimens of repaglinide were evaluated in 12 elderly subjects with type 2 diabetes. On day 1, following a 10-hour fast, subjects received a single 2-mg dose of repaglinide. Starting on day 2 and continuing for 7 days, each subject received a 2-mg dose of repaglinide 15 minutes before each of the three main meals. On day 9, subjects received a single 2-mg dose of repaglinide. Pharmacokinetic profiles, including area under the curve (AUC), log(AUC), maximal concentration (Cmax), log(Cmax), time to maximal concentration (Tmax), and half-life (T(1/2)), were determined at completion of the single- and multiple-dose regimens (days 1 and 9, respectively). Trough repaglinide values were collected on days 2 through 7. The mean log(AUC) values after multiple dosing were significantly higher than the values obtained after a single dose. The mean values for log(Cmax), and Tmax were comparable after each dosing regimen. The T(1/2) of repaglinide after multiple dosing was 1.7 hours. The trough values for repaglinide were low. No hypoglycemic events were reported. The pharmacokinetic profiles of repaglinide after single- and multiple-dose regimens were similar, and repaglinide was well tolerated by elderly subjects with type 2 diabetes.

D2-like dopamine receptors are not detectable on human peripheral blood lymphocytes.

The binding of [3H]-nemonapride to human peripheral blood lymphocytes (PBL) was studied using various competing ligands specific for D2-like dopamine receptors. There is no detectable stereoselectivity for the stereoisomers of butaclamol, and competitions with haloperidol and sulpiride also show no evidence of specific binding to D2-like dopamine receptors. RT-PCR of RNA from human lymphocytes showed that there is no detectable D2 mRNA (even with nested PCR). D3 mRNA was, however, detectable by RT-PCR, but only at low levels that could not be detected by Northern blots of PBL total RNA.

Pharmacokinetics of repaglinide in subjects with renal impairment.

OBJECTIVE: To evaluate the effect of renal impairment and renal failure on the pharmacokinetics and safety of repaglinide. METHODS: We conducted a phase I, multicenter, parallel-group, pharmacokinetic comparison trial with single and multiple doses of repaglinide in subjects with various degrees of renal impairment. Subjects with normal renal function (n = 6) and subjects with renal impairment (mild to moderate, n = 6; severe, n = 6) received treatment with 2 mg repaglinide for 7 days. Subjects in the hemodialysis group (n = 6) received two single doses of 2 mg repaglinide separated by a 7- to 14-day washout period. All subjects had repaglinide serum pharmacokinetic profiles measured for the first and last doses administered. Serum steady-state levels, urine levels, and dialysate levels were also measured. RESULTS: Pharmacokinetic parameters did not show significant changes after single or multiple doses of repaglinide, although the elimination rate constant in the group with severe renal impairment decreased after 1 week of treatment. Subjects with severe impairment had significantly higher area under the curve values after single and multiple doses of repaglinide than subjects with normal renal function. No significant differences in values for maximum serum concentration or time to reach maximum concentration were detected between subjects with renal impairment and those with normal renal function. Hemodialysis did not significantly affect repaglinide clearance. CONCLUSIONS: Repaglinide was safe and well tolerated in subjects with varying degrees of renal impairment. Although adjustment of starting doses of repaglinide is not necessary for renal impairment or renal failure, severe impairment may require more care when upward adjustments of dosage are made.

Characterisation of brain D2 dopamine receptors solubilised by lysophosphatidylcholine.

Brain D2 dopamine receptors have been solubilised using lysophosphatidylcholine. The inclusion of proteinase inhibitors during solubilisation enables a preparation to be obtained containing a high proportion of solubilised D2 receptors with pharmacological properties similar to those of membrane-bound D2 receptors.

Reconstitution of solubilised brain D2 dopamine receptors into phospholipid vesicles.

D2 dopamine receptors have been solubilised from bovine caudate nucleus using cholate/sodium chloride in the presence of soyabean phospholipid. Reconstitution of the receptors into soyabean phospholipid vesicles has been achieved by dialysis to remove detergent and salt. The receptors are truly reconstituted as judged by sedimentation, electron microscopy, heat stability and analysis on sucrose density gradients. The ligand-binding properties of the reconstituted receptors resemble those of the solubilised preparation.