Effects of crystal habit on the sticking propensity of ibuprofen-A case study.

This study demonstrates the effect of active pharmaceutical ingredient (API) particle habit on the sticking propensity of ibuprofen. Four diverse crystal habits with similar physico chemical properties are reported and the sticking propensity was found to increase with shape regularity. The surface energy of the extreme habits were shown to be different where particles that were more regular in shape exhibited surface energies of 9mJ/m2 higher than those that were needle-like in habit. Computational and experimental data reveals that the increase in surface energy of the regular shaped particles can be attributed to the increase in the specific (polar) component, which is due to greater presence of faces which contain the carboxylic acid functionality at the surface. The increase in the specific energy component is shown to correlate with the sticking propensity of ibuprofen. It is proposed that investigation of the chemical causality of sticking, for this API and others, using the techniques demonstrated in this paper will be of increasing importance.

Investigation of intracellular signalling cascades mediating stimulatory effect of a Gymnema sylvestre extract on insulin secretion from isolated mouse and human islets of Langerhans.

AIM: Traditional plant-based remedies such as Gymnema sylvestre (GS) extracts have been used to treat diabetes mellitus for many centuries. We have shown previously that a novel GS extract, OSA®, has a direct effect on insulin secretion but its mode of action has not been studied in detail Thus this study investigated the possible underlying mechanism(s) by which OSA® exerts its action. METHODS: The effects of OSA® on [Ca(2+)]i and K(+) conductances were assessed by Ca(2+) microfluorimetry and electrophysiology in dispersed mouse islets and MIN6 ß-cells, respectively. Isolated mouse (from 20 to 25 mice) and human (from 3 donors) islets, and MIN6 ß-cells, were used to investigate whether the stimulatory effect of OSA® on insulin secretion was dependent on the presence of extracellular calcium and protein kinase activation. RESULTS: OSA ®-induced insulin secretion from mouse islets and MIN6 ß-cells was inhibited by nifedipine, a voltage-gated Ca(2+) channel blocker, and by the removal of extracellular Ca(2+), respectively. OSA® did not affect the activities of KATP channels or voltage-dependent K(+) channels in MIN6 ß-cells but it caused an increase in intracellular Ca(2+) ([Ca(2+)]i) concentrations in Fura-2-loaded mouse islet cells. The insulin secretagogue effect of OSA® was dependent, in part, on protein kinase activation since incubating mouse or human islets with staurosporine, a general protein kinase inhibitor, resulted in partial inhibition of OSA®-induced insulin secretion. Experiments using permeabilized, Ca(2+)-clamped MIN6 ß-cells revealed a Ca(2+)-independent component action of OSA® at a late stage in the stimulus-response coupling pathway. OSA®-induced insulin secretion was unexpectedly associated with a decrease in intracellular cAMP levels. CONCLUSIONS: These data indicate that the GS isolate OSA® stimulates insulin secretion from mouse and human islets in vitro, at least in part as a consequence of Ca(2+) influx and protein kinase activation.

A comparison of the effects of veratridine on tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels in isolated rat dorsal root ganglion neurons.

The effects of veratridine have been compared on tetrodotoxin-sensitive (TTXS) and tetrodotoxin-resistant (TTXR) voltage-gated sodium channels (VGSC) in rat dorsal root ganglion neurons. Veratridine caused a dose-dependent decrease in the peak amplitude of both TTXR and TTXS VGSC currents. When exposed to 25 microM veratridine, TTXS currents but not TTXR currents developed a clear persistent component. The deactivation of both TTXS and TTXR currents was slowed, as evidenced by the appearance of slowly decaying tail currents in voltage clamp records, but the slowing of deactivation was nearly 100 times greater for TTXS than for TTXR currents. Properties of the veratridine-modified VGSCs, derived from an analysis of the slow tail currents, were similar for both TTXS and TTXR in that the V50 for activation and the reversal potential were shifted to more negative potentials than control currents and by a similar amount for each. The relatively fast decay of veratridine-modified TTXR tail currents reflects a faster dissociation of veratridine from TTXR than from TTXS VGSCs. This difference probably underlies the lack of effect of veratridine on TTXR VGSCs in cells that are not voltage-clamped and undermines its value as a chemical activator of putative NaV1.8 TTXR channels.

The effect of dibutyryl cAMP on tetrodotoxin-sensitive and -resistant voltage-gated sodium currents in rat dorsal root ganglion neurons and the consequences for their sensitivity to lidocaine.

Tetrodotoxin-sensitive (TTXS) sodium currents in dorsal root ganglia (DRG) neurons were enhanced by DcAMP applied acutely or by pre-treatment. Pre-treatment increased peak TTXS by 28%. This compared to the increase of tetrodotoxin-resistant sodium currents (TTXR) of 123%. In both cases the increase was associated with a hyperpolarizing shift in activation potentials. Slow inactivation was slower for both TTXR and TTXS in DcAMP treated neurons but rates of recovery from inactivation were not altered. Lidocaine blocked TTX-R with an IC(50) of 0.51+/-0.15mM (n=9) which was reduced to 0.14+/-0.05mM (n=8, P<0.05) in DcAMP treated cells. The sensitivity of TTX-S currents to lidocaine was not altered by DcAMP (control EC(50)=0.89+/-0.16mM, n=9; DcAMP EC(50)=0.73+/-0.19mM, n=6). It is concluded that TTXS currents in DRG are, like TTX-R currents, enhanced by cAMP but whilst the pharmacology of TTXR channels with respect to lidocaine is altered, that to TTXS channels is not.

The use of the rat isolated vagus nerve for functional measurements of the effect of drugs in vitro.

In this article we describe how to dissect, set up and use the rat isolated vagus nerve in a 'grease gap' apparatus which provides a simple and practical method for measuring the effects of drugs on the membrane potential of axons in the nerve in vitro. Some discussion of the origins and development of the technique as well as the strengths and disadvantages of the preparation as a neuropharmacological tool are included. The vagus nerve conducts action potentials in at least three distinct types of axons that can be measured extracellularly as compound action potentials and distinguished on the basis of their conduction velocity and excitability. Activity in myelinated A fibres and unmyelinated C fibres can be measured separately easily. The axons express receptors for a wide range of putative neurotransmitter agents including 5-HT, GABA and ATP as well as other agents such as capsaicin, anandamide, bradykinin and prostanoids. Responses to all of these chemicals can be measured as a depolarization of the nerve fibres. The vagus nerve is an important target for a wide range of drugs and the isolated preparation provides a fairly simple preparation for studying their effects. The isolated vagus nerve is also a convenient system in which the effects of drugs that have been discovered using heterologous expression systems can be assayed on receptors and ion channels that are expressed in a native neural system.

Differential sensitivity to tetrodotoxin and lack of effect of prostaglandin E2 on the pharmacology and physiology of propagated action potentials.

1. We have studied the effects of prostaglandin E(2) (PGE(2)) on action potential propagation in the isolated, desheathed vagus and saphenous nerves of rats using an extracellular grease gap recording method. 2. PGE(2) evoked a small depolarization of vagus nerves but had no effect on the stimulation threshold, size or latency of either the A wave (corresponding to conduction in A fibres) or the C wave (corresponding to conduction in C fibres) of the compound action potential (CAP) recorded from either vagus or saphenous nerves. 3. Lidocaine (0.01 - 10 mM) reduced all components of the CAP of both vagus and saphenous nerves. PGE(2) had no significant effect on the sensitivity of any component of the CAP to lidocaine. 4. Tetrodotoxin (TTX, 10 microM) blocked completely both the A wave and the C wave of the CAP in either vagus or saphenous nerves. 5. In saphenous nerve preparations the A wave was blocked by lower concentrations of TTX than the C wave or any component of the CAP in vagus nerve preparations which suggests that somatosensory A fibres express a different sub-type of TTX-sensitive voltage-gated sodium channel (VGSC) than somatosensory C-fibres or visceral sensory fibres. 6. Chemical activation of VGSCs with veratridine (10 or 50 microM) induced a depolarization in either nerve. The depolarization induced by 50 microM veratridine was blocked by 10 microM TTX. 7. Although TTX-insensitive VGSCs are expressed by some vagal and some somatosensory neurones they do not appear to be expressed functionally in the axons.

Indirect actions of bradykinin on neonatal rat dorsal root ganglion neurones: a role for non-neuronal cells as nociceptors.

1. In this study we have investigated the action of bradykinin (Bk) on cultured neonatal rat dorsal root ganglion (DRG) cells, with the aim of elucidating whether the neuronal response to Bk is influenced by association with non-neuronal satellite cells. 2. Bradykinin (100 nM) evoked an inward current (I(Bk)) in 51 of 58 voltage clamped DRG neurones (holding potential (V(h)) = -80 mV) that were in contact with non-neuronal satellite cells. 3. Bradykinin failed to evoke an inward current in isolated DRG neurones (V(h) = -80 mV) that were not in contact with non-neuronal satellite cells (n = 41). 4. The lack of neuronal response to Bk was not influenced by time in culture. Bradykinin failed to evoke a response in isolated neurones through 1-5 days in culture. By contrast neurones in contact with satellite cells responded to Bk throughout the same time period. 5. Failure of isolated neurones to respond to Bk was not due to the replating procedure or to selective subcellular distribution of receptors/ion channels to the processes rather than the somata of neurones. 6. Using Indo-1 AM microfluorimetry Bk (100 nM) was demonstrated to evoke an intracellular Ca(2+) increase (Ca(Bk)) in DRG neurones in contact with non-neuronal satellite cells and in isolated neurones. 7. These data suggest that the inward current response to Bk requires contact between DRG neurones and non-neuronal satellite cells. This implies an indirect mechanism of action for Bk via the non-neuronal cells, which may perform a nociceptive role. However, Bk can also act directly on the neurones, since it evokes Ca(Bk) in isolated neurones. The relationship between Ca(Bk) and the Bk-induced inward current is unknown at present.

Bradykinin evokes a Ca2+-activated chloride current in non-neuronal cells isolated from neonatal rat dorsal root ganglia.

We have studied the effect of bradykinin (Bk) on fibroblast-like satellite (FLS) cells isolated from cultures of neonatal rat dorsal root ganglia (DRG). In voltage-clamped FLS cells Bk evoked an inward current response that was concentration dependent with a half-maximal concentration of 2 nM. In indo-1 AM-loaded FLS cells Bk evoked a rise in intracellular Ca2+ that was concentration dependent with a half-maximal concentration of 1 nM. The FLS cells still produced an inward current in response to Bk in the absence of extracellular Ca2+ but the response was inhibited if the intracellular concentration of EGTA was increased from 0.5 to 5 mM, which suggests that the inward current was dependent on the release and subsequent rise of intracellular Ca2+. The reversal potential of the Bk-induced inward current was consistent with the current being due to an increase in Cl- conductance and shifted in a Nernstian manner when the intracellular Cl- concentration was reduced. The inward current response to Bk was blocked by the B2 receptor antagonist HOE-140, which indicates that the response was due to activation of B2 receptors. The data suggest that Bk evokes a rise in intracellular Ca2+ and activation of a Ca2+-activated Cl- conductance in the FLS cells and raise the possibility that FLS cells contribute to the pro-inflammatory effects of Bk in DRG.

One-way cross-desensitization between P2X purinoceptors and vanilloid receptors in adult rat dorsal root ganglion neurones.

1. Capsaicin and ATP can activate ligand-gated cation channels in nociceptive rat dorsal root ganglion (DRG) neurones. We have studied cross-desensitization between these two agents in rat isolated DRG neurones using the whole-cell voltage-clamp technique. 2. ATP (10 microM) activated an inward current in DRG neurones at a holding potential of -60 mV. ATP evoked 'fast' responses that underwent rapid activation and desensitization, 'slow' responses that activated and desensitized more slowly, or responses that displayed a mixture of these two characteristics. The time course of the response to ATP was not related obviously to capsaicin sensitivity. 3. Prior application of capsaicin (0.5 microM) increased the proportion of cells displaying only fast responses to ATP (10 microM) suggesting that cross-desensitization had occurred between capsaicin and the slow component of the ATP response. Prior desensitization to ATP had no apparent effect on the inward current response to capsaicin (0.5 microM). 4. Cross-desensitization between capsaicin and ATP was Ca2+ dependent. 5. Changing the membrane holding potential (Vh) to +40 mV for brief period before applying ATP at -60 mV had a similar effect to capsaicin, i.e. the proportion of cells displaying only fast responses to ATP was increased significantly. This effect of depolarization was not Ca2+ dependent. 6. The heterogenity of responses to ATP is probably due to co-expression of homomeric P2X3 receptors and heteromeric receptors comprising P2X3 subunits with other P2X subunits. We propose that the change in time course of the ATP response produced by prior desensitization to capsaicin is due to selective cross-desensitization with the heteromeric P2X receptors.

Evidence for novel cell cycle checkpoints in trypanosomes: kinetoplast segregation and cytokinesis in the absence of mitosis.

Trypanosoma brucei has a single nucleus and a single kinetoplast (the mitochondrial genome). Each of these organelles has a distinct S phase, which is followed by a segregation period, prior to cell division. The segregation of the two genomes takes place in a specific temporal order by interaction with microtubule-based structures, the spindle for nuclear DNA and the flagellum basal bodies for the kinetoplast DNA. We used rhizoxin, the anti-microtubule agent and polymerisation inhibitor, or the nuclear DNA synthesis inhibitor aphidicolin, to interfere with cell cycle events in order to study how such events are co-ordinated. We show that T. brucei cytokinesis is not dependent upon either mitosis or nuclear DNA synthesis, suggesting that there are novel cell cycle checkpoints in this organism. Moreover, use of monoclonal antibodies to reveal cytoplasmic events such as basal body duplication shows that some aphidicolin treated cells appear to be in G(1) phase (1K1N) but have activated some cytoplasmic events characteristic of G(2) phase (basal body segregation). We discuss a possible dominant role in trypanosomes for kinetoplast/basal body segregation in control of later cell cycle events such as cytokinesis

Molecular characterisation of cloned bradykinin B1 receptors from rat and human.

This report describes the characterisation of cloned rat and human bradykinin B1 receptors in African green monkey kidney fibroblast (Cos-7) cells. A ligand binding assay with [3H]des-Arg10-kallidin was used to compare their pharmacology with respect to known bradykinin B1 and B2 receptor ligands. In addition, the pharmacology of T-kinin and its' derivative des-Arg11-T-kinin was investigated. The cloned rat receptor had a similar pharmacology to that of the recently described mouse receptor and differs from that described for the human receptor. The rat receptor had a higher affinity for des-Arg11-T-kinin than the human receptor. These differences in pharmacological properties may relate to the presence of T-kinin, bradykinin and their des-Arg derivatives as the major physiological peptides in rat and the predominance of kallidin and its derivatives in human. We confirm that the rat bradykinin B1 receptor gene is organised in a two exon structure and differs from the human gene which has a three exon structure and we further examine the inducible expression of this gene in a wide range of tissues using Northern blotting.

A study of the voltage dependence of capsaicin-activated membrane currents in rat sensory neurones before and after acute desensitization.

1. Responses to capsaicin in isolated sensory neurones have been shown to desensitize in a Ca2+- and voltage-dependent manner. We have studied desensitization of capsaicin-activated currents in cultured adult rat dorsal root ganglion (DRG) neurones over a range of membrane potentials using whole-cell patch-clamp techniques. 2. Acute desensitization of responses to capsaicin (0.5 microM) was significantly less when the holding potential (Vh) was +40 mV rather than -60 mV. This was not due only to reduced Ca2+ entry as the response to capsaicin was desensitized by the same amount whether prior exposure to capsaicin was at -60 or +40 mV. The I-V relationship for capsaicin-induced current, determined using a voltage step protocol, was outwardly rectifying and during the acute phase of desensitization the degree of outward rectification increased. 3. Acute desensitization and the increase in outward rectification that accompanied desensitization were inhibited when cells were dialysed with the rapid Ca2+ chelator BAPTA. Addition of a pseudosubstrate inhibitor of the Ca2+-calmodulin-dependent enzyme calcineurin (CI, 100 microM) prevented the increase in outward rectification although it did not cause a significant decrease of acute desensitization. 4. Removal of external Ca2+ or Mg2+ did not reverse the increase in outward rectification of capsaicin-activated current after Ca2+-dependent desensitization had occurred. This indicates that a voltage-dependent block of the capsaicin-activated ion channel by Ca2+ or Mg2+ was not responsible for the observed changes in the properties of the capsaicin-activated conductance.

Examples of successful crystal structure prediction: polymorphs of primidone and progesterone.

The field of crystal structure prediction and its potential value to the pharmaceutical industry is described. The process of structure prediction employed here is summarized and the results of its application to primidone and progesterone are reported. It is shown that the process successfully generates the known polymorphs of these molecules, starting from the molecular structure alone. Observations related to the application of the structure prediction process are reported.

A comparison of the effects of capsaicin with inhibitory nerve stimulation in the rat anococcygeus muscle in vitro.

Capsaicin was used to test whether centrifugal activation of sensory fibres in the rat anococcygeus muscle can contribute to non-adrenergic non-cholinergic (NANC) relaxation of the muscle. In a solution containing 0.5 mM Ca2+ and in the presence of carbachol (10 microM) capsaicin evoked a fast concentration-dependent relaxation of the muscle that was usually followed by a smaller, slower, relaxant response. The fast relaxant response was reduced when extracellular Ca2+ was raised to 2.5 mM, desensitized after a single application of capsaicin and was blocked by tetrodotoxin (1 microM) or ruthenium red (10 microM). The fast response was greatly reduced by haemoglobin, by cold storage of the muscles or by N-monomethyl-L-arginine (100 microM) in the absence but not in the presence of L-arginine (100 microM). It is concluded that centrifugal activation of sensory fibres evokes a nitric oxide-mediated relaxation of the anococcygeus muscles that probably contributes to electrically evoked NANC relaxation.

Comparative, general pharmacology of SDZ NKT 343, a novel, selective NK1 receptor antagonist.

1. The in vitro and in vivo pharmacology of SDZ NKT 343 (2-nitrophenyl-carbamoyl-(S)-prolyl-(S)-3-(2-naphthyl)alanyl-N-benzyl-N- methylamide), a novel tachykinin NK1 receptor antagonist was investigated. 2. SDZ NKT 343 inhibited [3H]-substance P binding to the human NK1 receptor in transfected Cos-7 cell membranes (IC50 = 0.62+/-0.11 nM). In comparison, in the same assay Ki values for FK888, CP 99,994, SR 140,333 and RPR 100,893 were 2.13+/-0.04 nM, 0.96+/-0.20 nM, 0.15+/-0.06 nM and 1.77+/-0.41 nM, respectively. SDZ NKT 343 showed a markedly lower affinity at rat NK1 receptors in whole forebrain membranes (IC50 = 451+/-139 nM). 3. SDZ NKT 343 caused an increase in EC50 as well as reduction in the number of binding sites (Bmax) determined for [3H]-substance P, suggesting a non-competitive interaction at the human NK1 receptor. SDZ NKT 343 also caused a reduction in the maximum elevation of [Ca2+]i evoked by substance P (SP) in human U373MG cells and depressed the maximum [Sar9]SP sulphone-induced contraction of the guinea-pig isolated ileum. The antagonism of SP effects on U373MG cells by SDZ NKT 343 was reversible. 4. SDZ NKT 343 showed weak affinity to human NK2 and NK3 receptors in transfected Cos-7 cells (Ki of 0.52+/-0.04 microM and 3.4+/-1.2 microM, respectively). SDZ NKT 343 was inactive in a broad array of binding assays including the bradykinin B2 receptor the histamine H1 receptor, opiate receptors and adrenoceptors. SDZ NKT 343 only weakly inhibited the voltage-activated Ca2+ and Na+ currents in guinea-pig dorsal root ganglion neurones. The enantiomer of SDZ NKT 343, (R,R)-SDZ NKT 343 was about 1000 times less active at human NK1 receptors expressed in Cos-7 cell membranes. 5. Contractions of the guinea-pig ileum by [Sar9]SP sulphone were inhibited by SDZ NKT 343 in a concentration-dependent manner, with an IC50 = 1.60+/-0.94 nM, while the enantiomer (R,R)-SDZ NKT 343 was 100 times less active (IC50 = 162+/-26 nM). In comparison, in the same assay IC50 values for other NK1 receptor antagonists CP 99,994, SR 140,333, RPR 100,893 and FK 888 were 2.90+/-07 nM, 0.14+/-0.02 nM, 11.4+/-2.9 nM and 2.4+/-0.83 nM, respectively. 6. In anaesthetized guinea-pigs i.v. administered SDZ NKT 343 antagonized [Sar9]SP sulphone-evoked bronchoconstriction (70% reduction at 0.4 mg kg(-1), i.v.). Basal airway resistance, mean arterial blood pressure and heart rate were not affected. 7. In conclusion, SDZ NKT 343 is a highly selective NK1 receptor antagonist with high potency at the human and guinea-pig receptors. SDZ NKT 343 may be used as a potential novel therapeutic agent in human diseases where NK1 receptor hyperfunction is involved.

Capsazepine block of voltage-activated calcium channels in adult rat dorsal root ganglion neurones in culture.

1. We have found that capsazepine, a competitive antagonist at the vanilloid (capsaicin) receptor, blocks voltage-activated calcium currents in sensory neurones. 2. The block of calcium current was slow to develop with a half time of about one minute at 100 microM and lasted for the duration of the experiment. The rate of block of calcium current was strongly concentration-dependent. 3. The EC50 for the blocking effect at 0 mV was 7.7 +/- 1.4 microM after 6 min exposure to capsazepine. The EC50 at equilibrium was estimated to be 1.4 +/- 0.2 microM. 4. The block of calcium current showed some voltage-dependence but there was no indication of any selectivity of action for a calcium channel subtype. The characteristics of the blocking action of capsazepine on the residual current of cells which were pretreated with either omega-conotoxin or nimodipine were similar to control. 5. The data suggest that capsazepine, in addition to its competitive antagonism of vanilloid receptors, has a non-specific blocking action on voltage-activated calcium channels which should be taken into account when interpreting the effects of this substance on intact preparations in vitro or in vivo.

Chemical activators of sensory neurons.

Chemical activation of sensory neurons plays an important role in the somatosensory system. The actions of both endogenous mediators such as excitatory amino acids, acetylcholine, bradykinin, and ATP, as well as selective exogenous activators of nociceptive sensory neurons are reviewed. The physiological significance of these mediators in both nociception and other types of sensation are discussed.

A fluorescence in situ hybridisation study of the regulation of histone mRNA levels during the cell cycle of Trypanosoma brucei.

We have analysed the regulation of histone H2A, H2B, H4 and beta-tubulin RNA levels during the cell cycle of asynchronous cultures of Trypanosoma brucei by fluorescence in situ hybridisation. Whereas tubulin mRNA is detectable at high levels during the entire cell cycle, histone mRNA presence peaks during S phase and is not detectable during all other stages of the cell cycle within the sensitivity limits of this technique. We show that fluorescence in situ hybridisation can be used to characterise the distribution patterns of cell cycle regulated transcripts in asynchronous cell culture systems and discuss the possibilities and limitations of quantification of hybridisation patterns by means of computer-assisted image analysis.

PGE2 modulates the tetrodotoxin-resistant sodium current in neonatal rat dorsal root ganglion neurones via the cyclic AMP-protein kinase A cascade.

1. In current-clamp recordings, 1 microM prostaglandin E2 (PGE2) increased the excitability of neonatal rat dorsal root ganglion neurones. The current threshold for firing was reduced, and the response to a constant suprathreshold stimulation was modified such that a single evoked action potential was converted to a train of action potentials. The excitatory action of PGE2 was still apparent when action potentials were evoked in the presence of 500 nM tetrodotoxin. 2. In voltage-clamp experiments 1 microM PGE2 frequently increased the magnitude of the peak currents recorded, and caused a hyperpolarizing shift (of approximately 6 mV) in the activation curve for the tetrodotoxin-resistant sodium current (TTX-R INa). In some cells, the hyperpolarizing shift in the activation curve was accompanied by a decrease in peak conductance. PGE2 also caused a hyperpolarizing shift in the steady-state inactivation curve for the sodium current. 3. Extracellular application of the cAMP analogue dibutyryl cAMP (dbcAMP) at a concentration of 1 mM produced effects on both the current-voltage relationship and the steady-state inactivation curve for the TTX-R INa which were indistinguishable from those observed with PGE2. Prior exposure of the neurones to dbcAMP occluded the effect of a subsequent treatment with PGE2. 4. Forskolin (10 microM), a direct activator of adenylate cyclase, mimicked the effects of PGE2 and dbcAMP on TTX-R INa. The inactive congener of forskolin, 1, 9-dideoxyforskolin (10 microM), reduced the amplitude of TTX-R INa, but did not evoke a hyperpolarizing shift in the activation curve. 5. Intracellular perfusion of the neurones with an inhibitor of protein kinase A inhibited the effect of PGE2 on TTX-R INa. 6. PGE2 also reduced the amplitude of voltage-gated potassium currents (IK), which will contribute to the excitatory action. The mechanisms underlying the changes in IK have yet to be elucidated. 7. We propose that the PGE2-mediated increase in excitability in sensory neurones may be due, at least in part, to the cAMP-protein kinase A-dependent modulation of the tetrodotoxin-resistant sodium channel.

Modulation by calcineurin of 5-HT3 receptor function in NG108-15 neuroblastoma x glioma cells.

1. We have investigated the mechanism of regulation of 5-HT3 receptor channel sensitivity in voltage-clamped (-80 mV) NG108-15 neuroblastoma cells. 2. The 5-HT-induced inward current activated rapidly. The fast onset was followed by a biphasic decay which was characterized by two time constants, tau 1 (1.1 +/- 0.21s) and tau 2 (8.9 +/- 1.6s), respectively. Brief applications of 5-HT, applied at 2 min intervals, induced a decrease in the amplitude of the 5-HT3 receptor-mediated peak inward currents. 3. Buffering of intracellular calcium with the calcium chelator BAPTA (10 mM) instead of EGTA (10 mM) attenuated the 5-HT-induced loss of responsiveness of 5-HT3 receptors. Omission of calcium from the extracellular medium yielded a similar attenuation of loss of responsiveness. 4. Inclusion of the protein kinase inhibitor, staurosporine (1 microM) or of okadaic acid (1 microM), an inhibitor of protein phosphatases 1 and 2A, in the intracellular buffer solution did not affect 5-HT3 receptor sensitivity. 5. Injection of cyclosporin A-cyclophilin A complex (20 nM), which potently inhibits calcineurin, did not affect the time constants of the biphasic decay of the 5-HT response tau 1 (1.4 +/- 0.28s) and tau 2 (11.3 +/- 1.7s). The complex, however, prevented the loss of 5-HT3, receptor responsiveness upon repeated application of 5-HT. A similar, but weaker effect was observed after intracellular application of the autoinhibitory peptide domain of calcineurin (1 microM). 6. The recovery of desensitized 5-HT3 receptors upon a second application of 5-HT (1 microM) showed a half-life time (tau 1/2) of 2.6 +/- 0.12 min in control cells which was reduced to 1.6 +/- 0.09 min in cells treated with cyclosporin A-cyclophilin A (20 nM) complex. 7. We conclude that calcineurin does not affect the fast decay of the 5-HT3 receptor response but may be involved in a slower process which regulates channel activity.