A Gaussian-plume based Monte Carlo method for calculating radiation dose in the near field of buildings.

A Monte Carlo (MC) programme was written using the dose point kernel method to calculate doses in the roof zone of a building from nearby releases of radioactive gases. A Gaussian Plume Model (GPM) was parameterised to account for near-field building effects on plume spread and reflection from the roof. Rooftop recirculation zones and building-generated plume spread effects were accounted in a novel Dual Gaussian Plume (DGP) formulation used with the MC model, which allowed for the selection of angle of approach flow, plume release height in relation to the building and position of the release point in relation to the leading edge of the building. Three-dimensional wind tunnel concentration field data were used for the parameterisation. The MC code used the parameterised concentration field to calculate the contributions to effective dose from inhalation, cloud immersion from positron/beta particles, and gamma-ray dose for a wide range of receptor dose positions in the roof zone, including receptor positions at different heights above the roof. Broad trends in predicted radiation dose with angle of approach flow, release position in relation to the building and release height are shown. Alternative approaches for the derivation of the concentration field are discussed.

Conversion of simulated radioactive pollutant gas concentrations for a complex building array into radiation dose.

Methods used to convert wind tunnel and ADMS concentration field data for a complex building array into effective radiation dose were developed based on simulations of a site in central London. Pollutant source terms were from positron emitting gases released from a cyclotron and clinical PET radiotracer facility. Five years of meteorological data were analysed to determine the probability distribution of wind direction and speed. A hemispherical plume cloud model (both static and moving) was developed which enabled an expression of gamma-ray dose, taking into account build-up factors in air, in terms of analytic functions in this geometry. The standard building wake model is presented, but this is extended and developed in a new model to cover the concentration field in the vicinity of a roof top structure recirculation zone, which is then related to the concentration in the main building wake zone. For all models presented the effective dose was determined from inhalation, positron cloud immersion and gamma ray plume contributions. Results of applying these models for determination of radiation dose for a particular site are presented elsewhere.

Occupational radiation exposure during endovascular aortic procedures.

OBJECTIVES: To measure the radiation exposure of the operating team during endovascular aortic procedures, and to determine factors that predict high exposures. MATERIALS AND METHODS: Electronic dosimeters placed over and under protective lead garments, were used to prospectively record radiation exposure during endovascular aortic repairs performed in a designated interventional radiology suite. Univariate and multivariate linear regression analyses of predictors of radiation exposure were performed. RESULTS: A total of 26 infra-renal and 10 thoracic endovascular cases were studied. Median (IQR) patient age and body mass index were 76.0 (70.0-81.8) years and 26.2 (23.9-28.9) kg/m(2) respectively. Over-lead exposure to the operator was higher for thoracic than for infra-renal procedures (421.0 [233.8-597.8] µSv vs. 52.5 [27.8-179.8] µSv, p = .0003), reflecting a significant exposure to unprotected parts of the body. Under-lead exposures for operator and assistant were 5.5 (2.0-14.2) µSv and 1.0 (0.0-2.3) µSv respectively, which for an average caseload would comply with total body effective dose limits. Type of case and percentage of digital subtraction angiography (DSA) time in left anterior oblique angulations predicted dose to the operator (p < .0001). CONCLUSIONS: Thoracic procedures, DSA runs and obliquity of the C-arm are strong predictors of radiation exposure during endovascular aortic repairs. Understanding scatter radiation dynamics and instigating measures to minimise radiation exposure should be mandatory.

Predicting drug-induced changes in QT interval and arrhythmias: QT-shortening drugs point to gaps in the ICHS7B Guidelines.

BACKGROUND AND PURPOSE: The regulatory guidelines (ICHS7B) recommending inhibition of the delayed rectifier K(+) current (I(Kr)), carried by human ether-a-go-go-related gene (hERG) channels in cardiac cells (the hERG test), as a 'first line' test for identifying compounds inducing QT prolongation, have limitations, some of which are outlined here. EXPERIMENTAL APPROACH: hERG current was measured in HEK293 cells, stably transfected with hERG channels; action potential duration (APD) and arrhythmogenic effects were measured in isolated Purkinje fibres and perfused hearts from rabbits. KEY RESULTS: 576 compounds were screened in the hERG test: 58% were identified as hERG inhibitors, 39% had no effect and 3% were classified as stimulators. Of the hERG inhibitors, 92 were tested in the APD assay: 55.4% of these prolonged APD, 28.3% had no effect and 16.3% shortened APD. Of the 70 compounds without effect on hERG channels, 54.3% did not affect APD, 25.7% prolonged, while 20% significantly shortened APD. Dofetilide (hERG inhibitor; IC(50), 29 nM) prolonged QT and elicited early after-depolarizations and/or torsade de pointes (TdP) in isolated hearts. Mallotoxin and NS1643 (hERG current stimulators at 3 microM), levcromakalim and nicorandil (no effect on hERG current), all significantly shortened APD and QT, and elicited ventricular fibrillation (VF) in isolated hearts. CONCLUSION AND IMPLICATIONS: The hERG assay alone did not adequately identify drugs inducing QT prolongation. It is also important to detect drug-induced QT shortening, as this effect is associated with a potential risk for ventricular tachycardia and VF, the latter being invariably fatal, whereas TdP has an approximately 15-25% incidence of death.

Measurement of secretory vesicle pH reveals intravesicular alkalinization by vesicular monoamine transporter type 2 resulting in inhibition of prohormone cleavage.

1. The acidic interior of neuroendocrine secretory vesicles provides both an energy gradient for amine-proton exchangers (VMATs) to concentrate small transmitter molecules, for example catecholamines, and an optimal pH for the prohormone convertases which cleave hormone precursors. There is evidence that VMAT activity modulates prohormone cleavage, but in the absence of measurements of pH in secretory vesicles in intact cells, it has not been possible to establish whether these effects are attributable to raised intravesicular pH due to proton transport through VMATs. 2. Clones were generated of the hamster insulinoma cell line HIT-T15 expressing a pH-sensitive form of green fluorescent protein (GFP-F64L/S65T) targeted to secretory vesicles, with and without co-expression of VMAT2. In order to study prohormone cleavage, further clones were generated that expressed preprogastrin with and without co-expression of VMAT2. 3. Confocal microscopy of GFP fluorescence indicated that the pH in the secretory vesicles was 5.6 in control cells, compared with 6.6 in cells expressing VMAT2; the latter was reduced to 5.8 by the VMAT inhibitor reserpine. 4. Using a pulse-chase labelling protocol, cleavage of 34-residue gastrin (G34) was found to be inhibited by co-expression with VMAT2, and this was reversed by reserpine. Similar effects on vesicle pH and G34 cleavage were produced by ammonium chloride. 5. We conclude that VMAT expression confers the linked abilities to store biogenic amines and modulate secretory vesicle pH over a range influencing prohormone cleavage and therefore determining the identity of regulatory peptide secretory products.

Role of Ins(1,4,5)P3, cADP-ribose and nicotinic acid-adenine dinucleotide phosphate in Ca2+ signalling in mouse submandibular acinar cells.

cADP-ribose (cADPr) and nicotinic acid-adenine dinucleotide phosphate (NAADP) are two putative second messengers; they were first shown to stimulate Ca(2+) mobilization in sea urchin eggs. We have used the patch-clamp whole-cell technique to determine the role of cADPr and NAADP in relation to that of Ins(1,4,5)P(3) in mouse submandibular acinar cells by measuring agonist-evoked and second-messenger-evoked changes in Ca(2+)-dependent K(+) and Cl(-) currents. Both Ins(1,4,5)P(3) and cADPr were capable of reproducing the full range of responses normally seen in response to stimulation with acetylcholine (ACh). Low concentrations of agonist (10-20 nM ACh) or second messenger [1-10 microM Ins(1,4,5)P(3) or cADPr] elicited a sporadic transient activation of the Ca(2+)-dependent currents; mid-range concentrations [50-500 nM ACh, 50 microM Ins(1,4,5)P(3) or 50-100 microM cADPr] elicited high-frequency (approx. 2 Hz) trains of current spikes; and high concentrations [more than 500 nM ACh, more than 50 microM Ins(1,4,5)P(3) or more than 100 microM cADPr] gave rise to a sustained current response. The response to ACh was inhibited by antagonists of both the Ins(1,4,5)P(3) receptor [Ins(1,4,5)P(3)R] and the ryanodine receptor (RyR) but could be completely blocked only by an Ins(1,4,5)P(3)R antagonist (heparin). NAADP (50 nM to 100 microM) did not itself activate the Ca(2+)-dependent ion currents, nor did it inhibit the activation of these currents by ACh. These results show that, in these cells, both Ins(1,4,5)P(3)R and RyR are involved in the propagation of the Ca(2+) signal stimulated by ACh and that cADPr can function as an endogenous regulator of RyR. Furthermore, although NAADP might have a role in hormone-stimulated secretion in pancreatic acinar cells, it does not contribute to ACh-evoked secretion in submandibular acinar cells.

Ins(1,3,4,5)P4 is effective in mobilizing Ca2+ in mouse exocrine pancreatic acinar cells if phospholipase A2 is inhibited.

In enzymically isolated mouse pancreatic acinar cells, under conditions of whole-cell patch-clamp current recording, the effect of phospholipase C-coupled agonists can be mimicked by internal perfusion of the intracellular second messenger Ins(1,4,5)P3 (10 microM) or its analogue Ins(2,4,5)P3 (10 microM). The inositol trisphosphates mimic receptor activation by releasing Ca2+ from intracellular stores and by promoting Ca2+ influx across the surface membrane. This Ca(2+)-mobilizing role of inositol polyphosphates seems to be confined to the inositol trisphosphates because internal perfusion of Ins(1,3,4,5)P4 (10 microM) is not associated with any Ca(2+)-dependent current activation. In this study we investigate the effects of 4-bromophenacyl bromide (4BPB), a putative inhibitor of phospholipase A2 and arachadonic acid production, on inositol polyphosphate-induced Ca2+ signalling. At 10 microM, 4BPB has no effect on unstimulated Ca(2+)-dependent membrane currents. However, if 4BPB is applied to cells internally perfused with 10 microM Ins(1,4,5)P3 or Ins(2,4,5)P3 then the current responses are rapidly potentiated. In cells internally perfused with 10 microM Ins(1,3,4,5)P4, which has itself no effect on membrane currents, application of 4BPB resulted in the activation of Ca(2+)-dependent currents, seen either as repetitive spikes of current or as sustained current activations. The application of arachidonic acid blocks the current responses evoked by the inositol trisphosphates and by Ins(1,3,4,5)P4/4BPB. These results suggest that in enzymically isolated pancreatic acinar cells phospholipase A2 activity is exerting an inhibitory effect on inositol polyphosphate-mediated Ca2+ mobilization. 4BPB removes this inhibition and potentiates the responses to internally perfused inositol trisphosphates and, importantly, makes 10 microM Ins(1,3,4,5)P4 as effective as 10 microM Ins(1,4,5)P3 in mobilizing intracellular Ca2+ and in promoting Ca2+ influx.

Thapsigargin-induced Ca2+ mobilization in acutely isolated mouse lacrimal acinar cells is dependent on a basal level of Ins(1,4,5)P3 and is inhibited by heparin.

The tumour-promoting agent thapsigargin has been shown to inhibit the microsomal Ca(2+)-ATPase and cause Ca2+ mobilization in a variety of cell types including exocrine acinar cells [Bird, Obie and Putney (1992) J. Biol. Chem. 267, 18382-18386]. When applied to acutely isolated lacrimal acinar cells, thapsigargin caused a slow biphasic activation of both the Ca(2+)-dependent K+ and Cl- currents measured using the whole-cell patch-clamp technique. If the only action of thapsigargin is to inhibit sequestration into Ca2+ pools, then Ca2+ mobilization following exposure to thapsigargin indicates that there is a significant 'leak' of Ca2+ into the cytoplasm, which is normally countered by Ca(2+)-ATPase activity. In the present study, we introduced the Ins(1,4,5)P3 receptor antagonist heparin (200 micrograms/ml) into lacrimal acinar cells via the patch-clamp pipette. Following a 5 min preincubation in the presence of heparin, neither acetylcholine (1 microM) nor thapsigargin (1 microM) caused any significant increase in either Ca(2+)-dependent current. Caffeine has been shown to suppress basal Ins(1,4,5)P3 levels in exocrine acinar cells [Toescu, O'Neill, Petersen and Eisner (1992) J. Biol. Chem. 267, 23467-23470]. Preincubation with caffeine (10 mM) also inhibited the response to subsequent exposure to thapsigargin. These data suggest that, in acutely isolated lacrimal cells, the source of the Ca2+ leak which gives rise to Ca2+ mobilization following inhibition of Ca2+ re-uptake by thapsigargin is Ca2+ release, from Ins(1,4,5)P3-dependent Ca2+ pools, caused by resting Ins(1,4,5)P3 levels.

Acetylcholine- and caffeine-evoked repetitive transient Ca(2+)-activated K+ and C1- currents in mouse submandibular cells.

1. Resting and acetylcholine-induced membrane currents were measured in single mouse submandibular acinar cells using the patch-clamp whole-cell current recording technique. 2. Micromolar ACh activated a large, sustained outward, Ca(2+)-dependent K+ current and a single transient inward Ca(2+)-dependent C1-current. 3. Nanomolar ACh induced a series of transients in both the K+ and C1- currents; C1- current activation was now observed throughout the period of agonist application. We consider this repetitive transient current activation better able to support sustained fluid and electrolyte secretion than the response elicited by a high dose of agonist. 4. Repetitive K+ and C1- current transients were also induced by 1 mM-caffeine, consistent with caffeine-induced Ca2+ release from the Ca(2+)-sensitive Ca2+ stores which are thought to comprise part of the pathway for activation of secretion. 5. The ACh-induced current transients were inhibited by 10 mM-caffeine, 100 microM-IBMX and 10 microM membrane-permeable cyclic AMP. Therefore, it seems likely that caffeine is able to inhibit agonist-induced calcium mobilization via a cyclic AMP-dependent pathway.

Spatial and temporal distribution of agonist-evoked cytoplasmic Ca2+ signals in exocrine acinar cells analysed by digital image microscopy.

High resolution digital video imaging has been employed to monitor the spatial and temporal development of agonist-induced cytosolic Ca2+ signals in fura 2-loaded exocrine acinar cells. Enzymatically isolated mouse pancreatic and lacrimal acinar cells or small acinar cell clusters were used. These retain their morphological polarity so that the secretory granules in individual cells are located at one pole, the secretory pole. In acinar cell clusters the granules are located centrally, oriented to surround what would be in situ referred to as the lumen. In pancreatic and lacrimal acinar cells inositol-1,4,5-triphosphate-generating agonists [acetylcholine (ACh) and cholecystokinin octapeptide (CCK) for the pancreas and ACh in the lacrimal gland] give rise to a rapidly spreading Ca2+ signal that is initiated at the secretory pole of the cells. The initial increase in [Ca2+]i in the luminal pole is independent of extracellular Ca2+ indicating that the earliest detectable intracellular Ca2+ release is specifically located at the secretory pole. In lacrimal acinar cells ATP acts as an extracellular agonist, independent of phosphoinositide metabolism to activate a receptor-operated calcium influx pathway which, as for ACh, gives rise firstly to an increase in intracellular Ca2+ concentration in the secretory pole. We propose that this polar rise in intracellular Ca2+ concentration is due to Ca(2+)-induced Ca2+ release. By contrast, when Ca2+ release and Ca2+ influx are induced in the absence of receptor activation by thapsigargin and ionomycin, the Ca2+ signal develops diffusely and slowly with no localization to the secretory pole.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine-evoked increase in the cytoplasmic Ca2+ concentration and Ca2+ extrusion measured simultaneously in single mouse pancreatic acinar cells.

The intracellular free Ca2+ concentration ([free Ca2+]i) was measured simultaneously with the Ca2+ extrusion from single isolated mouse pancreatic acinar cells placed in a microdroplet of extracellular solution using the fluorescent probes fura-2 and fluo-3. The extracellular solution had a low total calcium concentration (15-35 microM), and acetylcholine (ACh), applied by microionophoresis, therefore only evoked a transient elevation of [free Ca2+]i lasting about 2-5 min. The initial sharp rise in [free Ca2+]i from about 100 nM toward 0.5-1 microM was followed within seconds by an increase in the total calcium concentration in the microdroplet solution ([Ca]o). The rate of this rise of [Ca]o was dependent on the [free Ca2+]i elevation, and as [free Ca2+]i gradually decreased Ca2+ extrusion declined with the same time course. Ca2+ extrusion following ACh stimulation was not influenced by removal of all Na+ in the microdroplet solution indicating that the Ca2+ extrusion is not mediated by Na(+)-Ca2+ exchange but by the Ca2+ pump. The amount of Ca2+ extruded during the ACh-evoked transient rise in [free Ca2+]i corresponded to a decrease in the total intracellular Ca concentration of about 0.7 mM which is close to previously reported values (0.5-1 mM) for the total concentration of mobilizable calcium in these cells. Our results therefore demonstrate directly the ability of the Ca2+ pump to rapidly remove the large amount of Ca2+ released from the intracellular pools during receptor activation.

The ATP-induced inward current in mouse lacrimal acinar cells is potentiated by isoprenaline and GTP.

1. ATP activates calcium (Ca2+) influx in mouse lacrimal acinar cells in the absence of phosphoinositide hydrolysis. Extracellular ATP (1 mM) activates receptor-operated cation channels, promoting entry of Na+ and Ca2+ (inward current). This Ca2+ influx in turn activates K+ channels resulting in a delayed, outward, current component. The present study uses patch-clamp current recording techniques to investigate the role of beta-adrenoceptor mechanisms, intracellular cyclic AMP and GTP in the regulation of the ATP-induced inward currents. 2. The beta-adrenoceptor agonist, isoprenaline (1 microM), does not increase the resting membrane currents but markedly enhances the ATP-induced inward and outward currents. This effect of isoprenaline is blocked by the beta-adrenoceptor antagonist propranolol. 3. Internal application of cyclic AMP mimics the potentiating effect of isoprenaline. 100 microM-cyclic AMP increases the ATP-induced inward and outward currents to about 200% as compared to control responses. 4. Pre-treatment of the cells with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX; 1 mM), also results in a marked potentiation of the ATP-induced inward currents to 170% as compared to control responses. 5. The ATP-induced inward current responses are not blocked by either the removal of extracellular Ca2+ or by chelation of intracellular Ca2+ (by inclusion of 10 mM-EGTA in the recording pipette). Both protocols did however block the potentiating effect of internal cyclic AMP on the ATP-induced inward current responses. 6. Intracellular ATP (10 mM) reduces the amplitude of the inward currents evoked by external ATP application by about 60% and the currents were no longer potentiated by internal cyclic AMP. 7. Intracellular GTP or GTP-gamma-S (100 microM in the pipette solution) potentiates the current responses to ATP, increasing both the amplitude and duration of the inward currents. 8. In excised inside-out patches, with ATP in the recording pipette (i.e. external ATP), the catalytic subunit of the cyclic AMP-dependent protein kinase activated the cation channels. The effect of the catalytic subunit was readily reversible and abolished by an inhibitor of the protein kinase. 9. External ATP activates Ca2+ influx in lacrimal acinar cells by a mechanism that is distinct from that activated by phosphoinositide-coupled receptors. The effect is mediated by direct activation of cation channels in the cell surface membrane which allow for significant entry of Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of alanine on insulin-secreting cells: patch-clamp and single cell intracellular Ca2+ measurements.

The effects of alanine, glucose and tolbutamide on insulin-secreting cells (RINm5F) have been investigated using patch-clamp and single cell intracellular Ca2+ measurements. When directly challenged with the amino acid L-alanine (2-10 mM) the cells underwent a sharp depolarization, which led to the generation of Ca2+ spike potentials and an increase in [Ca2+]i. The L-alanine-induced depolarization was associated with a net inward membrane current but no measurable change in the resistance of the cell. The latter effect was found to be in contrast to the actions of glucose (5-10 mM) and tolbutamide (100 microM), both of which depolarized cells and raised [Ca2+]i by an increase in the input resistance of the cell membrane, due to the closure of ATP-sensitive potassium channels. In the complete absence of external Na+ (by replacement with 140 mM NMDG+), L-alanine had no effects on either the membrane potential or [Ca2+]i. Similarly, replacing Na+ with NMDG+ in the continued presence of the amino acid resulted in a repolarization of the membrane and an attenuation of the L-alanine-induced rise in [Ca2+]i. The Na+ channel blocker TTX (1-2 microM) had no effects on the alanine-evoked electrical activity. Exchange of the L-form of the amino acid with the D-stereoisomer had similar actions to those of removing external Na+, since D-alanine had no effects on the membrane potential or [Ca2+]i. The actions of L-alanine were also found to be mimicked by the N-methylated amino acid analogue methylamino isobutyric acid (MeAIB) (2-10 mM), suggesting that the A-type electrogenic amino acid cotransport system operates in the RINm5F insulin-secreting cell line.

Extracellular ATP activates receptor-operated cation channels in mouse lacrimal acinar cells to promote calcium influx in the absence of phosphoinositide metabolism.

In exocrine acinar cells a variety of neurotransmitters (e.g. acetylcholine) stimulate phosphatidylinositol 4,5-bisphosphate hydrolysis elevating intracellular calcium to activate calcium-dependent membrane currents (outward K+ and inward Cl-). This study shows that in lacrimal acinar cells extracellular application of ATP is also associated with outward and inward current responses; these, however, are not the result of phosphoinositide metabolism. ATP directly activates receptor-operated cation channels which permit influx of Na+ and Ca+ (the inward current). The elevation in [Ca2+]i which results is sufficient to activate the outward K+ current. ATP thus promotes Ca+ influx in the absence of phosphoinositide metabolism.

Comparative study of the effects of cromakalim (BRL 34915) and diazoxide on membrane potential, [Ca2+]i and ATP-sensitive potassium currents in insulin-secreting cells.

Patch-clamp and single cell [Ca2+]i measurements have been used to investigate the effects of the potassium channel modulators cromakalim, diazoxide and tolbutamide on the insulin-secreting cell line RINm5F. In intact cells, with an average cellular transmembrane potential of -62 +/- 2 mV (n = 42) and an average basal [Ca2+]i of 102 +/- 6 nM (n = 37), glucose (2.5-10 mM): (i) depolarized the membrane, through a decrease in the outward KATP current, (ii) evoked Ca2+ spike potentials, and (iii) caused a sharp rise in [Ca2+]i. In the continued presence of glucose both cromakalim (100-200 microM) and diazoxide (100 microM) repolarized the membrane, terminated Ca2+ spike potentials and attenuated the secretagogue-induced rise in [Ca2+]i. In whole cells (voltage-clamp records) and excised outside-out membrane patches, both cromakalim and diazoxide enhanced the current by opening ATP-sensitive K+ channels. Diazoxide was consistently found to be more potent than cromakalim. Tolbutamide, a specific inhibitor of ATP-sensitive K+ channels, reversed the effects of cromakalim on membrane potential and KATP currents.

Vasopressin directly closes ATP-sensitive potassium channels evoking membrane depolarization and an increase in the free intracellular Ca2+ concentration in insulin-secreting cells.

The effects of arginine-vasopressin (AVP) (0.01-1 microM) on membrane potential, [Ca2+]i and ATP-sensitive potassium channels have been studied in the insulin-secreting cell line RINm5F. In whole cells, with an average spontaneous cellular transmembrane potential of -64 +/- 3 mV (n = 33) and an average basal [Ca2+]i of 102 +/- 6 nM (n = 40), AVP evoked: (i) membrane depolarization, (ii) voltage-dependent Ca2+ spike-potentials and (iii) a sharp rise in [Ca2+]i. Single-channel current events recorded from excised outside-out membrane patches show that AVP closes potassium channels that are also closed by tolbutamide (100 microM) and opened by diazoxide (100 microM). AVP acts on KATP channels specifically from the outside of the membrane and a soluble cytosolic messenger appears not to be involved, since there is no channel activation in cell-attached membrane patches when the peptide is added to the bath solution.

Inositol 1,3,4,5-tetrakisphosphate and inositol 1,4,5-trisphosphate act by different mechanisms when controlling Ca2+ in mouse lacrimal acinar cells.

In internally perfused single lacrimal acinar cells the competitive inositol 1,4,5-trisphosphate (Ins 1,4,5-P3)-antagonist heparin inhibits the ACh-evoked K+ current response mediated by internal Ca2+ and also blocks both the Ins 1,4,5-P3-evoked transient as well as the sustained K+ current increase evoked by combined stimulation with internal Ins 1,4,5-P3 and inositol 1,3,4,5-tetrakisphosphate (Ins 1,3,4,5-P4). When, during sustained stimulation with both Ins 1,4,5-P3 and Ins 1,3,4,5-P4, one of the inositol polyphosphates is removed, the K+ current declines; whereas removal of Ins 1,4,5-P3 results in an immediate termination of the response, removal of Ins 1,3,4,5-P4 only causes a very gradual and slow reduction in the current. Ins 1,3,4,5-P4 is therefore not an acute controller of Ca2+ release from stores into the cytosol, but modulates the release of Ca2+ induced by Ins 1,4,5,P3 by an unknown mechanism, perhaps by linking Ins 1,4,5 P3-sensitive and insensitive Ca2+ stores.

Inositol 1,3,4,5-tetrakisphosphate is essential for sustained activation of the Ca2+-dependent K+ current in single internally perfused mouse lacrimal acinar cells.

We have examined the effects of various inositol polyphosphates, alone and in combination, on the Ca2+-activated K+ current in internally perfused, single mouse lacrimal acinar cells. We used the patch-clamp technique for whole-cell current recording with a set-up allowing exchange of the pipette solution during individual experiments so that control and test periods could be directly compared in individual cells. Inositol 1,4,5-trisphosphate (Ins 1,4,5 P3) (10-100 microM) evoked a transient increase in the Ca2+-sensitive K+ current that was independent of the presence of Ca2+ in the external solution. The transient nature of the Ins 1,4,5 P3 effect was not due to rapid metabolic breakdown, as similar responses were obtained in the presence of 5 mM 2,3-diphosphoglyceric acid, that blocks the hydrolysis of Ins 1,4,5 P3, as well as with the stable analogue DL-inositol 1,4,5-trisphosphorothioate (Ins 1,4,5 P(S)3) (100 microM). Ins 1,3,4 P3 (50 microM) had no effect, whereas 50 microM Ins 2,4,5 P3 evoked responses similar to those obtained by 10 microM Ins 1,4,5 P3. A sustained increase in Ca2+-dependent K+ current was only observed when inositol 1,3,4,5-tetrakisphosphate (Ins 1,3,4,5 P4) (10 microM) was added to the Ins 1,4,5 P3 (10 microM)-containing solution and this effect could be terminated by removal of external Ca2+. The effect of Ins 1,3,4,5 P4 was specifically dependent on the presence of Ins 1,4,5 P3 as it was not found when 10 microM concentrations of Ins 1,3,4 P3 or Ins 2,4,5 P3 were used.(ABSTRACT TRUNCATED AT 250 WORDS)