Growth factor release from dentine matrix by pulp-capping agents promotes pulp tissue repair-associated events.

AIM: To characterize growth factor release from dentine by pulp-capping agents and to determine the effects of liberated dentine extracellular matrix (dECM) components on pulp cells in the key wound healing processes of migration and cell growth. METHODOLOGY: Powdered human dentine was exposed to solutions of calcium hydroxide, white and grey mineral trioxide aggregate (MTA) (ProRoot, (Dentsply Tulsa, Tulsa, OK, USA) over 14 days. The solubilized dECM components were dialysed and lyophilized and characterized using multiplex quantitative ELISA. Following dECM component extraction dentine was analysed using Fourier-transformed infrared spectroscopy (FTIR). Primary rat dental pulp cells (RDPCs) were exposed to dECM components (0.1-100 µg mL-1 ) released by calcium hydroxide, white and grey MTA, and cell growth and chemotactic responses were assessed. Statistical differences between the experimental and control groups were determined using one-way anova. RESULTS: A broad range of growth factors, many not previously reported in dentine, were liberated by these pulp-capping agents, including SCF, M-CSF, GM-CSF, IGFBP-1, NGF and GDNF. White and grey MTA liberated more growth factors than calcium hydroxide. FTIR analysis of dentine exposed to pulp-capping agents showed partial depletion of amide bands I, II and III, with little alteration in phosphate peaks compared to untreated dentine. dECM components released by white and grey MTA induced significantly more cell growth at low-to-moderate concentrations (P â‰¦ 0.05) examined in this study and significantly enhanced cell chemotaxis at all concentrations compared with controls (P â‰¦ 0.05). CONCLUSIONS: White and grey MTA solubilize a broad range of bioactive molecules from dentine, which can induce proliferation and chemotaxis in pulp cells.

Should pulp chamber pulpotomy be seen as a permanent treatment? Some preliminary thoughts.

AIM: To investigate the benefits of pulpotomy (to the level of the floor of the pulp chamber) as an endodontic treatment for teeth with vital pulps. METHODOLOGY: Seventeen patients, aged 7-54 years (mean of 37.2 year), were treated by pulpotomy and filling with ProRoot MTA(®) in premolar or molar teeth with vital pulps and without clinical evidence of irreversible pulpitis. The patients were then followed up for 12 to 24 months and the teeth then assessed by clinical and radiographic examination. Statistical analysis was performed with Kaplan-Meier survival probability statistics to estimate the survival of the treated teeth. RESULTS: At 24 months, the survival rate without any complementary treatment was estimated to be 82%. Two of the 17 treated teeth required root canal treatment for pain control and one for prosthetic reasons. CONCLUSIONS: Under the conditions of this study, pulpotomy offered a viable alternative to root canal treatment for teeth with vital pulps in the short term. However, there is insufficient clinical evidence to consider this technique for the treatment of every permanent tooth. Nevertheless, it should be considered as a potential alternative approach to be further developed for future applications.

Dentin-pulp complex regeneration: from lab to clinic.

Dentistry is entering an exciting era in which many of the advances in biotechnology offer opportunities for exploitation in novel and more effective therapies. Pulp healing is complex and dependent on the extent of injury, among many other factors. Many of the molecular and cellular processes involved in these healing events recapitulate developmental processes. The regulation of odontoblast activity is clearly central to pulp healing, and an understanding of the mechanisms involved in these processes is necessary to enable laboratory studies to be translated to clinic application. Transcriptome analysis has identified changes in many odontoblast genes during the life-cycle of this cell and its responses to injurious challenge. The p38 MAPKinase pathway appears to be central to the transcriptional control of odontoblasts and may provide a key target for therapeutic intervention. The many recent advances in knowledge of pulpal stem cells and molecular signaling molecules within the tooth, now provide exciting opportunities for clinical translation to novel therapies. Such translation will require the partnership of researchers and skilled clinicians who can effectively apply advances in knowledge to appropriate clinical cases and develop novel therapies which can be realistically introduced into the clinic.

Comparison of operative procedure variables on pulpal viability in an ex vivo model.

AIM: To measure and compare the responses of pulp tissue to cavity preparation and restoration variables using a novel tooth slice culture model. METHODOLOGY: Experimental cavities (265) were continuously cut, under carefully controlled conditions, into the dentine of the labial aspect of 28-day-old Wistar rat incisors, and slices of these teeth maintained in organ culture for up to 2 weeks. The experimental variables examined were: the preparation method, remaining dentine thickness, coolant, drill speed, conditioning with EDTA and filling materials. The reactions of the dentine-pulp complex to the experimental variables were measured using pathohistometric analysis and the correlations between variables were determined using analysis of variance statistical tests. RESULTS: In rank order of surgically induced restorative pulpal injury, from the most to the least injurious were: remaining dentine thickness, absence of coolant during cavity preparation, bur speed, cavity conditioning treatments and the filling material. CONCLUSIONS: To reduce pulp injury and to promote pulpal repair activity, the correct use of appropriate materials are important. However, of relatively greater importance is the operative technique adopted, the need to avoid the excess removal of dentine and to minimize trauma during preparation.

Ten-year outcome of root fillings in the General Dental Services in England and Wales.

AIM: To consider the survival of root canal treatment provided within the General Dental Services in England and Wales, with failure being defined as re-treating of a root canal, apical surgery or extraction. METHODS: A data set was established consisting of patients, 18 years or older, whose birthdays were included within a set of randomly selected dates and whose restoration records contained the placement of one or more direct restorations or crowns in courses of treatment between September 1990 and January 2002. The history of each root canal-treated tooth was consulted, and the next date for an intervention on the root canal of the tooth, defined as a re-treatment, apical surgery or extraction, was obtained. Thus, a data set was created of root canal-treated teeth, with the dates of root canal filling placement and the dates, if any, of re-intervention. RESULTS: Data for over 80,000 different adult patients were analysed, and a total of 30,843 root canal-treated teeth identified from the data over a period of 11 years. The proportion surviving without further treatment of the root canal was estimated at 74% within an observation time of up to 10 years, with survival being strongly correlated with the characteristics of the patient, including age and treatment history, with older patients having root canal treatment with earlier re-intervention than those of younger patients. CONCLUSION: Within the data set analysed, an estimated 74% of root canal-treated teeth pass through 10 years without re-treatment, apical surgery or extraction.

Root canal retreatment.

UNLABELLED: Root canal therapy is not always successful and an increasing number of patients are requesting retreatment to address intra-radicular infection. The armamentarium available to assist the dentist, some of which is described in this article, has never been greater. CLINICAL RELEVANCE: This paper discusses the causes of failure and reviews many of the techniques available to the practitioner to tackle the problem.

Limited proteolysis study of structure-function relationships in Sh I, a polypeptide neurotoxin from a sea anemone.

The structure-function relationships of the neurotoxic polypeptide Sh I, from the sea anemone Stichodactyla helianthus, have been studied using limited proteolysis with trypsin and endoproteinase Lys-C. Major products from each of the proteolytic digests were characterised using N-terminal peptide sequencing and amino-acid analysis or mass spectrometry. Of the six possible tryptic cleavage sites in Sh I, the bonds adjacent to Arg-13 and Lys-47 were found to be the most susceptible, complete cleavage occurring within minutes. Cleavages adjacent to Lys-32 and Lys-46 proceeded more slowly and cleavage adjacent to Arg-45 was the slowest. The sixth potential site, adjacent to Lys-4, was not cleaved at all. All derivatives were inactive as crustacean neurotoxins. Cleavage with endoproteinase Lys-C generated two major products. Derivatives cleaved adjacent to Lys-32 and either Lys-46 or Lys-47 were isolated. Both were inactive, indicating that either cleavage adjacent to Lys-32 or the removal of the C-terminal lysine residue(s) was sufficient to abolish activity. Lys-4 again was refractory to cleavage. The sequence of cleavage events correlated well with the static accessibility of the lysyl and arginyl side chains and to a lesser extent with the accessibility of the carbonyl oxygen of susceptible peptide bonds, as measured from the solution structure of Sh I determined by 1H-NMR. In the case of Lys-4, the lack of cleavage by trypsin and endoproteinase Lys-C may reflect a lack of flexibility in this region. The effects of the various cleavages on biological activity emphasise that the surface of the protein near the reverse turn encompassing Asp-6, Asp-7 and Glu-8 is essential for activity.

Analysis of the inotropic: chronotropic selectivity of dobutamine and dopamine in anaethetised dogs and guinea-pig isolated atria.

The previously reported in vivo inotropic selectivity of dobutamine and dopamine compared with isoprenaline has been demonstrated in anaesthetised bivagotamised open chest dogs. Responses of heart rate, right ventricular tension, left ventricular dP/dtmax, and blood pressure were measured. Compared with isoprenaline, dobutamine and dopamine were 1.8 and 2.4 times more active in increasing cardiac contractility than rate. The inotropic selectivity of dopamine but not that of dobutamine was abolished by pretreatment of dogs with syrosingopine. In guinea-pig isolated atria, isoprenaline, dobutamine, and dopamine were all slightly rate selective although this was less for dopamine. In atria incubated with phenoxybenzamine from guinea-pigs pretreated with reserpine only the dopamine dose-response curves were displaced to the right indicating a considerable indirect sympathomimetic component. The in vivo inotropic selectivity of dopamine could therefore be explained on the basis of this indirect activity being manifested at lower doses in the myocardium where the stores of catecholamines are more abundant than at the sinoatrial node. However, it is concluded that the inotropic selectivity of dobutamine seen in vivo is not due to indirect activity, reflex effects, or to a difference in the beta-adrenoceptors mediating the rate and tension responses of the heart. Possible alternative explanations are discussed.

Prevention of intra-coronary thrombosis in the anaesthetised dog: the importance of thromboxane A2 and thrombin.

Vapiprost (GR32191, a TxA2 antagonist), r-hirudin, aspirin, ticlopidine and aspirin plus ticlopidine were examined for their ability to prevent electrically-induced thrombosis in an artificially stenosed coronary artery in the anaesthetised dog. Drugs or vehicle were administered prior to a 2 h period of electrical damage which was followed by a further 2 h observation period. In all vehicle-treated animals, blood flow markedly declined with onset of the damaging current; 80% completely occluded. All treatments reduced the incidence of complete occlusion to a similar extent. Vapiprost and r-hirudin also largely prevented the decline in blood flow both during and following the damage period whilst aspirin and ticlopidine, either alone or in combination were much less effective. With r-hirudin treatment, marked cyclic changes in flow occurred throughout the experiment; these were abolished by administration of vapiprost. In this dog model, TxA2 and thrombin appear to work in concert to produce coronary thrombosis, ADP being of minor importance. The superior effect of vapiprost over aspirin suggests a beneficial role for endogenous prostacyclin.

Differential ability of agonists to express distinct pools of fibrinogen (GpIIb/IIIa) receptors which can mediate the aggregation of human platelets.

We have investigated the effect of two procedures that modify human platelet surface membrane glycoprotein (Gp) IIb and IIIa complexes upon whole blood platelet aggregation to a range of agonists. (A) Irreversible disruption of complexes by temporary (30 min) Ca2+-deprivation with EGTA at 37 degrees C. (B) Binding of a monoclonal antibody M148 to the complex. EGTA exposure abolished aggregation to ADP, adrenaline and PAF. In contrast, full aggregation curves to collagen and U-46619 could still be established. EGTA exposure reduced M148 binding to platelets by 80%. Excess M148 abolished aggregation to ADP, PAF, collagen and U-46619. However, upon removal of unbound antibody from platelets full aggregation curves to collagen and U-46619 but not to ADP and PAF could be re-established. Thus human platelet aggregation to ADP, PAF and adrenaline appears absolutely dependent upon surface membrane GpIIb/IIIa complexes. In contrast, collagen and U-46619 cause expression of an additional distinct pool of Gp complexes inaccessible to EGTA and M148 in unstimulated platelets which is intimately involved in aggregation to these agonists.

A specific thromboxane receptor blocking drug, AH23848, reduces platelet deposition on vascular grafts in man.

The antithrombotic effect of a specific thromboxane A2 receptor blocking drug, AH23848, on radio-labelled platelet deposition in mature Dacron aorto-bifemoral grafts has been evaluated in patients. Thirty patients were randomly allocated to AH23848 70 mg, aspirin 300 mg plus dipyridamole 75 mg or placebo 8-hourly for 9 days. AH23848 inhibited platelet aggregation induced by the thromboxane A2 mimetic U-46619; no such effect was observed with aspirin plus dipyridamole. 111In-platelet uptake was measured as the thrombogenicity index (TI) which is a measure of the daily rate of accumulation of platelets by the graft. The mean (s.e. mean) value of 0.193 (0.029) on placebo was significantly reduced to 0.115 (0.022) by AH23848 (p less than 0.05) but only to 0.175 (0.028) by aspirin plus dipyridamole. There was no difference in mean platelet life span between the three treatment groups. The pronounced antithrombotic effect of AH23848 implicates thromboxane A2 in the process of platelet deposition in arterial prostheses and demonstrates the considerable promise of thromboxane receptor blocking drugs as antithrombotic therapy.

AH6809, a prostaglandin DP-receptor blocking drug on human platelets.

1. The effect of AH6809 (6-isopropoxy-9-oxoxanthene-2-carboxylic acid) has been studied upon the anti-aggregatory and aggregatory actions of various agents on human platelets in whole blood. 2. Prostaglandin D2 (PGD2), BW245C, 9 alpha, 11 beta-PGF2, PGI2 and 5'-N-ethylcarboxamide adenosine (NECA) all inhibited ADP-induced platelet aggregation in whole blood. The anti-aggregatory activity of PGD2, BW245C and 9 alpha, 11 beta-PGF2 but not PGI2 or NECA was antagonized by AH6809. NECA was antagonized by AH6809. 3. The antagonism of the anti-aggregatory activity of PGD2 by AH6809 was concentration-related and could be overcome by increasing the concentration of PGD2. Analysis of the data yielded an apparent pA2 for AH6809 of 5.35. 4. At approximately 10 fold higher concentrations than those required to antagonize the action of PGD2, AH6809 also antagonized the aggregatory effect of U-46619 in whole blood (pA2 = 4.45). However, concentrations of AH6809 up to 300 microM were without effect upon either ADP- or platelet activating factor (Paf)-induced aggregation (pA2 less than 3.5). 5. The potency of AH6809 against PGD2 and U-46619 was increased in a resuspended platelet preparation suggesting that the drug is extensively bound to plasma proteins. However, in resuspended platelets the specificity of AH6809 relative to that seen in whole blood was reduced since aggregation by ADP and Paf was also slightly antagonized. 6. In conclusion, AH6809 appears to be a weak but specific DP-receptor blocking drug on human platelets and should prove to be a useful drug tool for defining the involvement of endogenous PGD2 in platelet aggregation and classifying the mode of action of anti-aggregatory prostanoids.

Effects of hypoxia, elevated K+ and acidosis on the potency of verapamil, diltiazem and nifedipine in the guinea-pig isolated papillary muscle.

1 The effects of the structurally diverse calcium channel blockers verapamil, nifedipine and diltiazem were investigated on the force of contraction of guinea-pig, electrically stimulated papillary muscles in vitro. 2 Calcium channel blocking potency was assessed either as a direct negative inotropic effect or as the ability of the drugs to antagonise the positive inotropic effects of added calcium (Ca2+). By either method, the rank order of potency was found to be nifedipine greater than verapamil greater than diltiazem. 3 Various factors which mimic some of the consequences of acute ischaemia in vivo, namely low pH, hypoxia and elevated K+, in combination, but not singly, enhanced the negative inotropic potency of verapamil and to lesser extent that of diltiazem, but not that of nifedipine. 4 Whilst the various interventions, especially in combination, produced a profound negative inotropic effect themselves, this was not responsible per se for the potentiation of the negative inotropic effects of verapamil and diltiazem, since the negative inotropic effects of nifedipine and dinitrophenol were not potentiated under the 'ischaemic' conditions. 5 By use of antagonism of the positive inotropic action of exogenous Ca2+ as an alternative measure of potency, the differential influence of 'ischaemia' on calcium channel blocker potency was confirmed. The effect of verapamil was potentiated some 9 fold, that of diltiazem about 2 fold, and that of nifedipine was unchanged. 6 The differential effect of 'ischaemia' on the potencies of the calcium channel blockers was unexpected. Verapamil (but not nifedipine) is thought to bind to the calcium channel at a specific site not easily accessible from the extracellular space. 'Ischaemic' conditions may cause membranal perturbations which allow verapamil easier access to its binding site thus increasing its negative inotropic potency.

Selective reserpine-induced supersensitivity of the positive inotropic and chronotropic responses to isoprenaline and salbutamol in guinea-pig isolated atria.

1 Dose-response curves for the positive inotropic and chronotropic responses to isoprenaline were obtained in atria from untreated guinea-pigs and those receiving various reserpine pretreatments. 2 Tension responses were unaffected, whereas rate responses were depressed by the lowest dose of reserpine (0.05 mg/kg i.p. at 24 hours). 3 With larger 24 h doses and a 3 day pretreatment, the rate and tension dose-response curves were progressively displaced to the left, indicating supersensitivity which was greater for tension at each pretreatment. 4 No supersensitivity to histamine or Ca2+ could be detected, leading to the conclusion that it was selective for the beta-adrenoceptor agonists possibly at the receptor level. 5 As an indication of the adrenergic neurone depleting effectiveness of each reserpine dosage, preparations were exposed to test doses of beta-phenylethylamine. 6 Salbutamol was a partial agonist in untreated atria, the maximum rate (63.3%) and tension (10.0%) responses being less than those for isoprenaline. In atria from reserpine pretreated animals the supersensitivity was revealed as an increase of this maximum compared with isoprenaline. 7 The significance of this observation in relation to the possible mechanism of the supersensitivity is discussed.

Characteristics of the binding of [3H]-GR32191 to the thromboxane (TP-) receptor of human platelets.

1. The interaction of the specific thromboxane (TP-) receptor blocking drug, [3H]-GR32191 with human intact platelets and platelet membranes has been investigated in vitro. 2. On intact platelets, association of specific [3H]-GR32191 binding at 37 degrees C was biphasic, with an initial rapid component and a slower secondary phase. Dissociation experiments indicated displacement from two sites with t1/2 values of 8.1 and 65.6 minutes. Kd values derived from the kinetic rate constants for the rapid onset/offset and slow onset/offset phases were 0.4 and 0.5 nM respectively. 3. Competition binding of [3H]-GR32191 and GR32191 on intact platelets gave an IC50 of 2.3 nM. Scatchard analysis indicated a single class of binding site with a Kd of 2.2 nM. Further analysis of the data yielded a Hill slope of -1.0 again indicating an interaction at a single binding site. Saturation binding experiments gave a similar estimate of the Kd value for [3H]-GR32191 to that obtained from competition binding experiments. A possible explanation for the biphasic interaction of the GR32191 in intact platelets may lie in restriction of its access to and egress from a population of TP-receptors. 4. In platelet membranes at 37 degrees C, specific [3H]-GR32191 binding was complete within 5 min with a calculated association rate constant of 3.2 x 10(8) M-1 min-1. Dissociation of [3H]-GR32191 was relatively slow, with measurable specific binding persisting for > 40 min. Analysis of these data yielded a t1/2 of 17.7 min and a dissociation rate constant of 0.04 min-1 and indicated dissociation from a single site. The ti for dissociation appeared to be related to the contact time of platelet membranes with [3H]-GR32191.Derivation of a Kd from the kinetic rate constants gave a value of 0.13 nM.5. Competition binding of [3H]-GR32191 and GR32191 to platelet membranes gave an IC50 value of 3.5 nM. Scatchard analysis of these data indicated a single binding site with a Kd of 2.1 nM. Saturation binding experiments with [3H]-GR32191 yielded similar IC50 and Kd values to those from competition experiments.6. In further competition binding experiments, the TP-receptor agonists U-46619, STA2, EP171 and 9,1 1-azo PGH2 and antagonists SQ29,548, BM 13.177 and EP092 all competed with specific [3H]-GR32191 binding on intact platelets and, where determined, on platelet membranes. All compounds fully displaced specific [3H]-GR32191 binding. However, where tested, the ICso values for a particular compound were always greater when [3H]-GR32191 was the radioligand than when [3H]-SQ29,548 was used. At the concentrations used in these studies (2 and 5 nM respectively), platelets appeared to bind approximately twice as much [3H]-GR32191 as [3H]-SQ29,548.7. In conclusion, the interaction of [3H]-GR32191 with human intact platelets was complex but the data were consistent with an action at a single class of binding site; from competition experiments this appears to be the functional TP-receptor. The interaction of the drug with this binding site is, however,characterized by a slow dissociation. This characteristic was confirmed in studies with platelet membranes and does not therefore appear to be an artefact of diffusion. Estimates of the Kd of the drug differed depending on the method of determination. Because of the slow dissociation of [3H]-GR32191,those relying upon equilibrium of the radioligand with competing agent may be unreliable. The rate of dissociation also appeared to be related to the contact time of drug with receptor. An explanation for this phenomenon may lie in the ability of GR32191 to induce a change in the conformational state or location of the human platelet TP-receptor.

Reduction in the number of thromboxane receptors on human platelets after exposure to GR32191.

1. Exposure of human resuspended platelets in vitro for 30 min to the potent thromboxane A2 (TP)-receptor blocking drug GR32191, followed by its removal by dilution-dissociation, reduced the degree of subsequent binding to 2 nM [3H]-GR32191 by almost 50%. Exposure for longer periods (60 min) led to a further reduction. However, no change in the Kd of the radioligand was observed. 2. This effect of GR32191 could not be explained by persistent binding of drug to platelets since a dilution-dissociation stage, designed to remove all drug, was included prior to measurement of binding. 3. Using an alternative TP-receptor radioligand, [3H]-SQ29,548, to monitor receptor number, a reduction in Bmax was observed after GR32191 pre-treatment; the Kd value of the radioligand remained unchanged. 4. The effect was not a common property of TP-receptor blocking drugs since pre-exposure of platelets in vitro for 30 min to BM13.177 or SQ29,548 did not produce a fall in subsequent Bmax to [3H]-SQ29,548. 5. While the mechanism behind this apparent down-regulation of platelet TP-receptor is unknown, it may explain the long duration of action of GR32191 upon platelets in man which persists in the absence of detectable drug in the plasma.

GR32191, a highly potent and specific thromboxane A2 receptor blocking drug on platelets and vascular and airways smooth muscle in vitro.

1. The thromboxane A2 (TP)-receptor blocking activity and specificity of action of GR32191 ([1R-[1 alpha(Z),2 beta,3 beta,5 alpha]]-(+)-7-[5-([1,1'-biphenyl] -4-ylmethoxy)-3-hydroxy-2-(1-piperidinyl)cyclopentyl]-4-heptoni c acid has been evaluated in human platelets and various smooth muscle preparations, both vascular and non-vascular, from a range of species including man. 2. Utilising a platelet counting method to assess aggregation the drug was found to antagonise, in a surmountable manner, human platelet aggregation produced by the TP-receptor agonists, U-46619, EP171 and SQ26655, in whole blood and physiological buffer, with pA2 values of approximately 8.3 and 8.7 in the two media respectively. In the presence of GR32191 the rate of aggregation induced by U-46619 was slowed. 3. The effect of GR32191 upon U-46619-induced platelet shape change and aggregation in platelet-rich plasma was evaluated utilising a turbidometric technique. Both shape change and aggregation were antagonised by GR32191. At relatively high concentrations of the drug a slowing of aggregation and shape change to U-44619 was seen and an unsurmountable antagonism became apparent. 4. The action of GR32191 upon human platelets was specific with platelet aggregation induced by adenosine 5'-diphosphate, platelet activating factor, vasopressin and adrenaline and the inhibitory effects of prostacylin (PGI2), prostaglandin D2 (PGD2) and N-ethylcarboxamide-adenosine (NECA) being unaffected by concentrations of the drug as high as 10 microM. Furthermore, at concentrations of up to 100 microM, the drug itself produced no shape change or aggregation, of human platelets. 5. GR32191 also specifically and potently antagonised in a competitive, surmountable manner the contractile actions of U-46619 upon human vascular smooth muscle and antagonised U-46619-induced contractions of vascular and airways smooth muscle preparations from rat, dog, guinea-pig and rabbit with varying potency. This is discussed in terms of possible heterogeneity of TP-receptors. 6. GR32191 therefore represents a highly potent and specific TP-receptor blocking drug. This profile of action, coupled to its long duration of effect in man described elsewhere, make it an ideal drug tool for elucidating the physiological and pathophysiological role of thromboxane A2.

Thromboxane (Tx) A2 receptor blockade and TxA2 synthase inhibition alone and in combination: comparison of anti-aggregatory efficacy in human platelets.

1. The present study has compared the relative anti-aggregatory effect of various compounds which interfere with thromboxane (Tx) A2-dependent aggregation of human platelets in whole blood in vitro. These included the cyclo-oxygenase inhibitor aspirin, the TxA2 synthase inhibitor dazoxiben, the TxA2 (TP-) receptor blocking drug GR32191 and two compounds, R.68070 ((E)-5-[[[(3-pyridinyl) [3-(trifluoromethyl)phenyl]-methylen] amino]oxy] pentanoic acid) and CV-4151 [E)-7-phenyl-7-(3-pyridyl)-6-heptenoic acid), which possess both TP-receptor blocking and TxA2 synthase inhibitory activities in the same molecule. 2. GR32191, R.68070 and CV-4151 all antagonized aggregation to the TxA2 mimetic U-46619, with pA2 values of approximately 8.2, 5.4 and 4.8 respectively. This effect was specific, platelet aggregation induced by adenosine 5'-diphosphate (ADP) being unaffected by concentrations up to 10, 1000 and 300 microM respectively. In contrast, neither aspirin nor dazoxiben exhibited any measurable TP-receptor blocking activity. 3. The rank order of potency (pIC50) for inhibition of TxA2 formation in serum was R.68070 (7.4) greater than CV-4151 (6.9) greater than dazoxiben (5.7) greater than aspirin (5.3). In addition, all four drugs abolished collagen-induced platelet TxA2 formation. In contrast, GR32191 produced no consistent inhibition of TxA2 formation in either system up to concentrations of 10-30 microM. 4. The specificity of R.68070, CV-4151 and dazoxiben for TxA2 synthase was indicated by their ability to increase serum levels of prostaglandin E2 (PGE2) and PGD2 in parallel with decreases in TxA2 formation. This profile was not observed with aspirin or GR32191. However, high concentrations of R.68070 (100,microM) and CV-4151 (1000 microM) necessary for maximum TP-receptor blocking activity, produced substantially smaller increases in PGE2 and PGD2, consistent with an aspirin-like effect of these compounds upon cyclo-oxygenase. With dazoxiben (1000 microM), PGE2 and PGD2 levels remained elevated. 5. Aspirin inhibited collagen-induced platelet aggregation, the effect correlating with inhibition of TxA2 formation. Dazoxiben, whilst also achieving maximal inhibition of TxA2 formation, produced significantly less inhibition of aggregation than aspirin. In contrast, GR32191 (0.1-1O microM), at concentrations specific for TP-receptor blockade, produced a significantly greater antagonism of collagen-induced platelet aggregation than aspirin. This additional effect of GR32191 was absent in platelets pretreated with aspirin, indicating the probable involvement of an endogenous anti-aggregatory cyclo-oxygenase product in response to collagen stimulation. 6. R.68070 and CV-4151 also inhibited collagen-induced aggregation, with very high concentrations of R.68070 (100 microM) producing an effect equivalent to that of GR32191. 7. In contrast, the combination of GR32191 with either dazoxiben, R.68070 or CV-4151, at concentrations specific for TxA2 synthase, produced a synergistic inhibitory effect upon collagen-induced platelet aggregation which was greater than that achieved with either aspirin or any of the compounds used alone. Pretreatment of platelets with aspirin reversed this synergistic effect, consistent with it being dependent upon the formation and action of anti-aggregatory prostaglandins. 8. In conclusion, the present study has confirmed the superior platelet inhibitory profile of a combination of a TP-receptor blocking drug and a TxA2 synthase inhibitor to that of either activity alone. However, the maximum inhibitory effect of the currently available compounds, R.68070 and CV4151, which possess both activities in the same molecule, appears to be no greater in vitro than that obtained with the potent TP-receptor blocking drug, GR32191. This most probably reflects the inhibition by R.68070 and CV-4151 of platelet cyclo-oxygenase at the concentrations required for effective TP-receptor blockade which results in a reduction in the formation of anti-aggregatory prostanoids.

Comparison of the actions of U-46619, a prostaglandin H2-analogue, with those of prostaglandin H2 and thromboxane A2 on some isolated smooth muscle preparations.

1 The actions of the prostaglandin H2 (PGH2) analogue, U-46619, have been compared with those of PGH2 on thromboxane A2 (TxA2) on a range of isolated smooth muscle preparations in a superfusion cascade system. 2 U-46619 was a potent agonist on guinea-pig lung strip, dog saphenous vein and rat and rabbit aortae. In contrast, U-46619 was weak or inactive on guinea-pig ileum and fundic strip, cat trachea and dog and cat iris sphincter muscles, preparations on which either PGE2 or PGF2 alpha was the most potent agonist studied. 3 PGH2 was active on all of the preparations and displayed little selectivity. On some of the preparations, the actions of PGH2 may have been mediated indirectly by conversion to other prostanoids. 4 In contrast, TxA2 displayed the same pattern of selectivity as U-46619, being a potent agonist on the lung strip and vascular preparations but weak or inactive on the others. 5 It is suggested that U-46619 is a selective TxA2-mimetic and that it should therefore be a valuable tool in the study of the actions of TxA2.

Tooth slice organ culture for cytotoxicity assessment of dental materials.

The purpose of this work was to develop a tooth slice organ culture method to assess the response of the cells of the dental pulp to commonly used dental materials and products. Wistar rat tooth slices were grown in culture for two and ten days in the presence of dental materials. After culture, the tooth tissues were processed and the responses of the pulpal cells were analysed histomorphometrically. Cytotoxic cell destruction was observed following the direct application of test materials to tooth slices (n = 298) after 10 days in culture (MANOVA, P = 0.0001), whilst the restoration of prepared deep dentine cavities (n = 30), with test products, did not result in a significant amount of pulpal injury (MANOVA, P = 0.287). In rank order of causing pulpal injury, the test materials from the most to the least cell destructive, was; Salicylic acid. Calcium hydroxide, Kalzinol zinc oxide eugenol, high-mercury Amalgam, Prime & Bond, Dycal, Barium sulphate, Hypocal, Scotchbond, Calasept, Life and One-step. Tooth slice organ culture, provided a cytotoxicity screening method for dental materials, bearing a closer physiological resemblance to the clinical situation than cell culture screening methods. Tooth slice culturing may have the potential to replace some types of in vivo animal experimentation, as there is a clear need to reduce this form of testing.