Phase Ib dose-escalation study of the hypoxia-modifier Myo-inositol trispyrophosphate in patients with hepatopancreatobiliary tumors.

Hypoxia is prominent in solid tumors and a recognized driver of malignancy. Thus far, targeting tumor hypoxia has remained unsuccessful. Myo-inositol trispyrophosphate (ITPP) is a re-oxygenating compound without apparent toxicity. In preclinical models, ITPP potentiates the efficacy of subsequent chemotherapy through vascular normalization. Here, we report the results of an unrandomized, open-labeled, 3 + 3 dose-escalation phase Ib study (NCT02528526) including 28 patients with advanced primary hepatopancreatobiliary malignancies and liver metastases of colorectal cancer receiving nine 8h-infusions of ITPP over three weeks across eight dose levels (1'866-14'500 mg/m2/dose), followed by standard chemotherapy. Primary objectives are assessment of the safety and tolerability and establishment of the maximum tolerated dose, while secondary objectives include assessment of pharmacokinetics, antitumor activity via radiological evaluation and assessment of circulatory tumor-specific and angiogenic markers. The maximum tolerated dose is 12,390 mg/m2, and ITPP treatment results in 32 treatment-related toxicities (mostly hypercalcemia) that require little or no intervention. 52% of patients have morphological disease stabilization under ITPP monotherapy. Following subsequent chemotherapy, 10% show partial responses while 60% have stable disease. Decreases in angiogenic markers are noted in ∼60% of patients after ITPP and tend to correlate with responses and survival after chemotherapy.

INO-4995 therapeutic efficacy is enhanced with repeat dosing in cystic fibrosis knockout mice and human epithelia.

Progressive lung damage in cystic fibrosis (CF) has been linked to inadequate airway mucosal hydration. We previously demonstrated that an inositol tetrakisphosphate analog, 1-O-octyl-2-O-butyryl-myo-inositol 3,4,5,6-tetrakisphosphate octakis(propionoxymethyl)ester (INO-4995), regulates airway secretory and absorptive processes, affecting mucosal hydration by prolonged (24 h) inhibition of Na(+) and fluid absorption in CF human nasal epithelia (CFHNE). The objectives of this study were to further assess clinical potential of INO-4995 in CF through ascertaining in vivo activity in mice with CF, determining the effects of repeated administration on potency and determining cytoplasmic half-life. Uptake and metabolism of [(3)H]INO-4995 was monitored with HPLC to calculate intracellular half-life. INO-4995 was administered in vitro repeatedly over 4 to 8 days to CFHNE. Fluid absorption was assessed by blue dextran exclusion, and basal short-circuit current was measured in Ussing chambers. INO-4995 (1-100 microg/kg) was dosed intranasally either as a single dose or once per day over 4 days to gut-corrected CF mice. [(3)H]INO-4995 was rapidly taken up by epithelial cultures and converted to the active drug, which had a half-life of 40 hours. Repeated daily application of INO-4995 to CFHNE lowered the effective concentration for inhibition of fluid absorption and amiloride-sensitive short-circuit current in cultured CFHNE, and reduced nasal potential difference to nearly control levels in gut-corrected CF mice. Ca(2+)-activated Cl(-) channel activity was also boosted in cultures. Mouse nasal levels fell from abnormal levels to within 2 muA of normal with repeated exposure to 0.8 microg/kg over 4 days. These data support further development of INO-4995 for the treatment of CF.

myo-Inositol trispyrophosphate: a novel allosteric effector of hemoglobin with high permeation selectivity across the red blood cell plasma membrane.

myo-Inositol trispyrophosphate (ITPP), a novel membrane-permeant allosteric effector of hemoglobin (Hb), enhances the regulated oxygen release capacity of red blood cells, thus counteracting the effects of hypoxia in diseases such as cancer and cardiovascular ailments. ITPP-induced shifting of the oxygen-hemoglobin equilibrium curve in red blood cells (RBCs) was inhibited by DIDS and NAP-taurine, indicating that band 3 protein, an anion transporter mainly localized on the RBC membrane, allows ITPP entry into RBCs. The maximum intracellular concentration of ITPP, determined by ion chromatography, was 5.5×10(-3) M, whereas a drop in concentration to the limit of detection was observed in NAP-taurine-treated RBCs. The dissociation constant of ITPP binding to RBC ghosts was found to be 1.72×10(-5) M. All data obtained indicate that ITPP uptake is mediated by band 3 protein and is thus highly tissue-selective towards RBCs, a feature of major importance for its potential therapeutic use.

Inhibition of vascular calcification by inositol phosphates derivatized with ethylene glycol oligomers.

Myo-inositol hexakisphosphate (IP6) is a natural product known to inhibit vascular calcification (VC), but with limited potency and low plasma exposure following bolus administration. Here we report the design of a series of inositol phosphate analogs as crystallization inhibitors, among which 4,6-di-O-(methoxy-diethyleneglycol)-myo-inositol-1,2,3,5-tetrakis(phosphate), (OEG2)2-IP4, displays increased in vitro activity, as well as more favorable pharmacokinetic and safety profiles than IP6 after subcutaneous injection. (OEG2)2-IP4 potently stabilizes calciprotein particle (CPP) growth, consistently demonstrates low micromolar activity in different in vitro models of VC (i.e., human serum, primary cell cultures, and tissue explants), and largely abolishes the development of VC in rodent models, while not causing toxicity related to serum calcium chelation. The data suggest a mechanism of action independent of the etiology of VC, whereby (OEG2)2-IP4 disrupts the nucleation and growth of pathological calcification.

Roles of PIP2 in the membrane binding of MIM I-BAR: insights from molecular dynamics simulations.

In order to probe the roles of PIP2 in the interactions between MIM I-BAR and model membranes, we performed a series of 10 µs-scale coarse-grained molecular dynamics simulations. Our results indicate that PIP2 plays predominant roles in the membrane binding of MIM I-BAR in a concentration-dependent manner and via electrostatic interactions. Besides, we find that the occurrence of the membrane curvature may induce the re-distribution of lipids in the membrane and result in the local enrichment of PIP2 at negatively curved membrane areas. Combining these roles of PIP2 in the membrane binding of MIM I-BAR helps explain how MIM I-BAR senses negative curvature and, thus, contributes to maintaining membrane protrusions.

Inhibition of the phosphatidylinositol 3-kinase/Akt pathway by inositol pentakisphosphate results in antiangiogenic and antitumor effects.

The purpose of this study was to investigate the antiangiogenic and in vivo properties of the recently identified phosphatidylinositol 3-kinase (PI3K)/Akt inhibitor Inositol(1,3,4,5,6) pentakisphosphate [Ins(1,3,4,5,6)P5]. Because activation of the PI3K/Akt pathway is a crucial step in some of the events leading to angiogenesis, the effect of Ins(1,3,4,5,6)P5 on basic fibroblast growth factor (FGF-2)-induced Akt phosphorylation, cell survival, motility, and tubulogenesis in vitro was tested in human umbilical vein endothelial cells (HUVEC). The effect of Ins(1,3,4,5,6)P5 on FGF-2-induced angiogenesis in vivo was evaluated using s.c. implanted Matrigel in mice. In addition, the effect of Ins(1,3,4,5,6)P5 on growth of ovarian carcinoma SKOV-3 xenograft was tested. Here, we show that FGF-2 induces Akt phosphorylation in HUVEC resulting in antiapoptotic effect in serum-deprived cells and increase in cellular motility. Ins(1,3,4,5,6)P5 blocks FGF-2-mediated Akt phosphorylation and inhibits both survival and migration in HUVEC. Moreover, Ins(1,3,4,5,6)P5 inhibits the FGF-2-mediated capillary tube formation of HUVEC plated on Matrigel and the FGF-2-induced angiogenic reaction in BALB/c mice. Finally, Ins(1,3,4,5,6)P5 blocks the s.c. growth of SKOV-3 xenografted in nude mice to the same extent than cisplatin and it completely inhibits Akt phosphorylation in vivo. These data definitively identify the Akt inhibitor Ins(1,3,4,5,6)P5 as a specific antiangiogenic and antitumor factor. Inappropriate activation of the PI3K/Akt pathway has been linked to the development of several diseases, including cancer, making this pathway an attractive target for therapeutic strategies. In this respect, Ins(1,3,4,5,6)P5, a water-soluble, natural compound with specific proapoptotic and antiangiogenic properties, might result in successful anticancer therapeutic strategies.

High-affinity neurotensin receptor is involved in phosphoinositide hydrolysis stimulation by carbachol in neonatal rat brain.

Ontogenetic studies indicate that inositol phosphate accumulation in rodent brain tissue by cholinergic muscarinic agonists as well as expression of high-affinity neurotensin receptor (NTS1) peak at 7 days after birth. Herein, potential participation of this receptor in such effect was investigated. Cerebral cortex prisms of 7-day-old rats were preloaded with [3H]myoinositol and later incubated during 60 or 20 min in the presence of muscarinic agonist carbachol plus neurotensin and SR 48692, a non-peptide NTS1 antagonist. In 60-min incubation experiments, inositol phosphate accumulation by 10(-3) M carbachol was roughly 320%, an effect which remained unaltered plus 10(-6) M to 10(-4) M neurotensin but partially decreased with equimolar SR 48692 concentration. In 20-min incubation experiments, inositol phosphate accumulation by 10(-3) M carbachol was circa 240%, a value which attained 320-360% plus 10(-7) M neurotensin; this effect was totally blocked by 10(-7) M SR 48692. It was concluded that in inositol phosphate accumulation by carbachol, besides the cholinergic muscarinic receptor, the NTS1 receptor is likewise involved; findings at 60 min are attributable to the effect of endogenous neurotensin whereas those at 20 min most likely involve both endogenous and exogenously added peptide.

Impairment of glycosyl-phosphatidylinositol-dependent insulin signaling system in isolated rat hepatocytes by streptozotocin-induced diabetes.

The addition to different types of cells of an inositol-phosphate glycan, generated by the phospholipase C-catalyzed hydrolysis of a insulin-sensitive glycosyl-phosphatidylinositol (glycosyl-PI), mimics some of the biological effects of this hormone. Recently, a specific, time-, dose-, and energy-dependent transport system for this inositol-phosphate glycan has been identified in isolated rat hepatocytes. Here, we show that streptozotocin-induced diabetes mellitus reduced (by about 60%) the basal content of the insulin-sensitive glycosyl-PI in isolated rat hepatocytes. Moreover, streptozotocin-induced diabetes blocked the hydrolysis of the glycosyl-PI in response to insulin, diminished inositol phosphate-glycan uptake by the hepatocytes, and abolished the stimulatory effect of this compound on glycogen synthesis. All these metabolic changes caused by streptozotocin administration were reversed by treatment of the animals with insulin. Our results support the hypothesis that insulin resistance in streptozotocin-induced diabetic rats is related to the impairment of glycosyl-PI metabolism.

Insulin-like growth factor-I regulates cell proliferation in the developing inner ear, activating glycosyl-phosphatidylinositol hydrolysis and Fos expression.

The role of insulin-like growth factors (IGF) was investigated during the early development of the inner ear. IGF-I stimulated growth of otic vesicles that were isolated and cultured in vitro. IGF-I induced DNA synthesis, increased cell number, and mitotic rate in a dose-dependent manner at concentrations between 0.1-10 nM. IGF-II also induced growth but with a lower potency, whereas insulin had no effect. In the presence of IGF-I, otic vesicles developed from stage 18 to stage 21 in 24-h cultures, mimicking the normal mitotic pattern and morphogenesis in vivo. IGF-I also stimulated growth in the cochleovestibular ganglion. Binding of 125I-IGF-I to specific receptors occurred with high affinity. An autoradiographic study of sections from otic vesicles showed radiolabeled IGF-I in the epithelium. Immunoreactivity to IGF-I was detected in the otic vesicle and in the cochleovestibular ganglion. Intracellular signaling mechanisms of IGF were explored by studying the turnover of glycosylated phosphatidylinositols and the expression of Fos oncoprotein. IGF-I rapidly increased Fos levels in cultured otic vesicles. Furthermore, antisense oligonucleotides complementary to c-fos were able to inhibit IGF-I-induced growth. Both IGF-I-induced cell proliferation and Fos expression were blocked by an antiinositol phosphoglycan (alpha-IPG) antibody. This work suggests that IGF-I may be a candidate to regulate proliferative growth of the otic primordium during normal development and that this action requires the sequential modulation of glycosyl-phosphatidylinositol turnover and Fos expression.

Inositol trisphosphate isomers in angiotensin II-stimulated adrenal glomerulosa cells.

The stimulation of phosphoinositide metabolism by angiotensin II (Ang II) was studied in [3H]inositol-labelled bovine adrenal glomerulosa cells. After separation of the phosphoinositols by ion-exchange high-performance liquid chromatography, it was shown that the formation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) followed distinct kinetics. The first compound to increase upon stimulation with 10(-7) M Ang II was Ins(1,4,5)P3, which reached a maximum (250% of basal level) within 10 s. At lower concentrations of Ang II, this response was slower. The formation of Ins(1,4,5)P3 depended upon the concentration of Ang II, with an EC50 of 2.4 +/- 1.5 X 10(-9) M Ang II. The potency of Ang II in stimulating the turnover of phosphoinositides and in increasing the biosynthesis of aldosterone was very similar, whereas the peptide was ten times more potent in its ability to mobilize Ca2+. Ang II was also able to stimulate the production of Ins(1,4,5)P3 in permeabilized glomerulosa cells. This effect was mimicked by a non-hydrolysable analog of GTP (GTP gamma S), suggesting that a GTP binding protein is involved in the mechanism coupling the Ang II membrane receptor to phospholipase C. These results strengthen the view that Ins(1,4,5)P3 plays a key role as second messenger in the steroidogenic response to Ang II in adrenal glomerulosa cells.

Alpha-trinositol reduces edema formation at the site of scald injury.

BACKGROUND: The effects of alpha-trinositol (1D-myo-inositol-1,2,6-triphosphate, IP3) on burn-induced edema formation were investigated. METHODS: Lymph flow (QL; microliter/min) and lymph-to-plasma protein ratio (CL/CP) were monitored in groups of five to six dogs before and 4 hours after (1) a 5-second 100 degrees C or 90 degrees C foot paw scald; (2) IP3 (45 mg/kg intravenous bolus, then a 20 mg/kg/hr infusion) 30 minutes before or after 100 degrees C scald, or 30 minutes after 90 degrees C scald. Hind paw venous pressure was elevated and maintained by outflow restriction until reaching steady state QL and (CL/CP)min. Macromolecular reflection coefficient (1-CL/CP) was measured. Fluid filtration coefficient (Kf; ml/min/mm Hg/100 gm) was calculated. Relative paw weight gain (%) was measured. RESULTS: Compared with preburn values, scald uniformly produced significant increases in QL, CL/CP, and Kf, IP3 significantly (p < 0.02, ANOVA) reduced paw weight gain when given before, but not after, 100 degrees C burn (41% +/- 5% versus 18% +/- 7% preburn IP3 and 31% +/- 3% postburn IP3). Compared with 90 degrees C burn animals, postburn treatment significantly (p < 0.017) attenuated 4-hour increases in QL (550 +/- 87 versus 252 +/- 29 microliters/min), Kf (0.016 +/- 00 versus 0.007 +/- 00 microliter/min/mm/Hg/100 gm), and relative paw weight gain (28% +/- 3% versus 12% +/- 5%). CONCLUSIONS: alpha-Trinositol given after a 90 degrees C scald blunted edema formation at the site of scald, likely through reduced transmembrane fluid flux.

alpha-Trinositol decreases lung edema formation after smoke inhalation in an ovine model.

Inhalation injury is a dominant cause of mortality in thermally injured individuals. After acute lung injury induced by smoke inhalation, lung lymph flow (QL) increased and pulmonary microvascular reflection coefficient to protein (sigma) decreased. alpha-Trinositol (PP56, 1D-myo-inositol 1,2,6-trisphosphate) can decrease edema formation after thermal injury. We therefore tested the hypothesis that alpha-trinositol could decrease the pulmonary edema noted with inhalation injury. Seven days after surgical preparation, sheep were insufflated with smoke from burning cotton towels. The alpha-trinositol group (n = 8) were treated with alpha-trinositol (2 mg/kg + 3.5 mg.kg-1 x h-1). The sham group (n = 7) received an equal volume of 0.9% NaCl. The sham group showed a large increase in QL (9.3 +/- 1.7 to 54.1 +/- 8.8 ml/h) and a decrease in sigma (0.79 +/- 0.03 to 0.48 +/- 0.03) 24 h after smoke inhalation. alpha-Trinositol attenuated the increase in QL (8.1 +/- 1.2 to 25.6 +/- 6.9 ml/h) and the decrease in sigma (0.76 +/- 0.03 to 0.60 +/- 0.03) noted with smoke inhalation. alpha-Trinositol thus decreased the changes in pulmonary microvascular permeability and transvascular fluid flux noted with inhalation injury.

Competitive inhibition of phosphoinositide hydrolysis by the muscarinic receptor antagonist AF-DX 116 is of low affinity in mouse cerebral cortex.

Carbamylcholine stimulated [3H]inositol phosphate accumulation in mouse cerebral cortical slices with an ED50 value of approximately 70 microM. Increasing concentrations of the M2 selective muscarinic cholinergic receptor antagonist, AF-DX 116 (0.3-3.0 microM). produced parallel shifts to the right for concentration-response curves to carbamylcholine. A pA2 value for AF-DX 116 of 6.5 (low affinity) was obtained from Schild plot analysis. It is concluded that the M2 muscarinic receptor subtype, as defined by high affinity [3H]AF-DX 116 radioligand binding, is not appreciably coupled to polyphosphoinositide hydrolysis in the mouse cerebral cortex.

Pharmacological properties of peptides derived from an antibody against the tachykinin NK1 receptor for the neuropeptide substance P.

Two peptides were derived from the structural analysis of a previously described monoclonal antibody [Mol. Immunol. 37 (2000) 423] against the tachykinin NK(1) receptor for the neuropeptide substance P. Here we show that these two peptides were able to inhibit the inositol phosphate transduction pathway triggered both by substance P and neurokinin A, another high-affinity endogenous ligand for the tachykinin NK(1) receptor. They also reduced the cAMP production induced by substance P. By contrast, only one antagonist peptide was able to prevent substance P and neurokinin A from binding the receptor, as revealed both by biochemical and autoradiographic studies. First, these results illustrate the generality of the antibody-based strategy for developing new bioactive peptides. Second, they indicate that antagonists, even exhibiting very close amino acid composition, can interact with the tachykinin NK(1) receptor at different contact sites, some of them clearly distinct from the contact domains for endogenous agonists.

Age-related changes in muscarinic cholinoceptor function in guinea-pig and rat airways.

The influence of age on muscarinic cholinoceptor-mediated contraction and inositol phosphate accumulation was examined in rat and guinea-pig isolated tracheal tissue. Significant age-related changes in the sensitivity of guinea-pig tracheal tissue to acetylcholine, but not carbachol, were observed in the early maturation phase of growth with a 3.3-fold increase in potency between birth and 2 weeks of age followed by a 3.2-fold fall in potency between 2 and 12 weeks of age. Further ageing did not significantly change the potency of acetylcholine or carbachol. Whilst acetylcholinesterase inhibition caused a significant increase in acetylcholine potency, this was independent of animal age. In rat isolated tracheal tissue, contractile responses to both acetylcholine and carbachol remained unchanged with respect to animal age. Significant age-related decreases in inositol phosphate accumulation were observed in response to carbachol in the guinea-pig and rat and to acetylcholine in guinea-pig but not rat isolated tracheal tissue. This study has demonstrated significant age-related changes in the responsiveness of isolated tracheal tissue to carbachol and acetylcholine which were also species-specific.

Sequential enzymatic synthesis and biodistribution of radiolabelled inositol and inositol analogs.

The desired radiolabelled inositol (Ins) and inositol-1-phosphate (IP) were obtained from radiolabelled glucose by sequential enzyme reactions in a short time. Rapid separation and fractionation of enzymes and labelled products from the reaction mixture was achieved by HPLC with a gel-permeation chromatography column. Examination of the biodistribution of radiolabelled Ins, IP and their acetylated analogs suggested that intact Ins labelled with 11C would be more effective than 11C-labelled acetylated Ins as a brain diagnostic agent for positron emission tomographic studies of the metabolism of phosphatidylinositol and its role as a second messenger in the brain.

A membrane-permeant, bioactivatable derivative of Ins(1,3,4)P3 and its effect on Cl(-)-secretion from T84 cells.

The synthesis of rac-2,5,6-tri-O-butyryl-myo-inositol 1,3,4-trisphosphate hexakis(acetoxymethyl) ester [Bt3-Ins(1,3,4)P3/AM, 1], a membrane-permeant derivative of myo-inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] is reported. 1 inhibited calcium-mediated chloride secretion of T84 cells, suggesting a regulatory link of Ins(1,3,4)P3 and the biosynthesis of the known inhibitor myo-inositol 3,4,5,6-tetrakisphosphate.

Effects of angiotensin-II on inositol phosphate accumulation and calcium influx in bovine adrenal and Y-1 tumor adrenal cells.

The present study compared the action of angiotensin II (A-II) in bovine adrenal fasciculata cells (BA) and Y-1 adrenal tumor cells which are sensitive and resistant respectively to its steroidogenic effect. In both models, A-II induced a time and dose-dependent inositol phosphate (Ins-Ps) accumulation and calcium influx. However, in Y-1 cells the Ins-Ps production was low and only Ins-P1 and Ins-P2 were accumulated. The calcium influx in BA cells was observed after 15 seconds and remained linear as long as the hormone was present, whereas in Y-1 cells calcium influx started prior to 15 seconds and reverted to basal values after 45 seconds. The effects of A-II on both cell types were specific since they were blocked by A-II antagonists. Taken together these results demonstrate the presence of functional A-II receptors in both cell types which are coupled to the two main intracellular messenger systems. Thus, the A-II steroidogenic refractoriness of Y-1 cells is probably related to some alteration(s) located beyond the calcium and/or protein kinase C A-II-messenger system.

Inositol phosphates and phosphoinositides in rat liver nodules.

Total amounts and turnover rates of phosphoinositides and inositol phosphates in normal rat liver and hepatocyte nodules were investigated. Male Wistar rats were injected i.p. with [3H]inositol 18-20 h before killing. The amount of phosphatidylinositol in a homogenate preparation was roughly doubled in the nodules, though levels of polyphosphoinositides were approximately the same. Basal levels of inositol phosphates were the same in nodules and in normal liver. Turnover rates of inositol tris- and tetrakisphosphates were studied after stimulation of intact cells with vasopressin for different periods of time (0-5 min). The initial rate of formation of inositol trisphosphate after agonist exposure was fast in both nodular and normal cells. Nodular cells reached peak amount of inositol trisphosphate at 2.5-fold basal levels after 20 s, while normal cells peaked after 40 s at 4.5 times the basal amount. The level of inositol tetrakisphosphate was enhanced very quickly in normal cells, but in the nodular cells there was no increase of this inositol phosphate after vasopressin stimulation. To investigate the mechanism of this difference, the activities of inositol 1,4,5-trisphosphate kinase and of inositol 1,4,5-trisphosphate phosphatase were studied. Both activities were rapid and equal in nodules and normal liver. The amount of cell surface receptors for vasopressin was shown to be one-third in the nodules, as compared to normal cells. This quantitative decrease in receptor number was reflected in lower formation of inositol trisphosphate when stimulated with vasopressin, but could not explain the loss of inositol tetrakisphosphate response in nodules. The significance of the reported alterations in second messenger traffic for the growth regulation of nodular cells and for their progression to carcinoma is not yet known, but could add to the nodules being less dependent on growth regulating signals.

The effect of intracellular alkalinisation on intracellular Ca(2+) homeostasis in a human chondrocyte cell line.

Intracellular pH (pH(i)) is a well-established determinant of cartilage matrix metabolism. Changes to chondrocyte pH(i), and therefore matrix turnover rates, arise following joint loading. It is not yet clear whether pH changes exert their effects on matrix metabolism directly, or by changing the concentration of another, as yet unidentified, intracellular factor. In this study the effect of intracellular alkalinisation on intracellular [Ca(2+)] has been examined using the human chondrocyte C-20/A4 cell line. pH(i) was manipulated by the addition of weak bases to suspensions of chondrocytes and fluorimetric techniques were employed to measure pH(i) and [Ca(2+)](i). The effect of pH(i) changes on intracellular inositol 1,4,5-trisphosphate (IP(3)) levels was also determined. The pH-sensitive properties of the Ca(2+)-sensitive fluoroprobe employed in this study, Fura-2, were investigated such that artefactual effects of pH changes upon the dye could be discounted. It was demonstrated that, for dye loaded into cells, alkalinisation resulted in a small increase in the affinity of the dye for Ca(2+) ions. Intracellular alkalinisation elicited by treatment with either of the weak bases trimethylamine or ammonium chloride initiated a rise in [Ca(2+)](i). This effect was too large to be explicable by the effects of pH changes on Fura-2 and was not dependent on the presence of extracellular Ca(2+) ions. Prior depletion of intracellular Ca(2+) stores by treatment with thapsigargin inhibited alkalinisation-induced increases in [Ca(2+)](i) and intracellular alkalinisation was also associated with increased levels of intracellular IP(3). These results confirm that alkaline pH(i) changes associated with dynamic loading of cartilage also result in knock-on alterations to [Ca(2+)](i). Given the sensitivity of cartilage matrix metabolism to [Ca(2+)](i) it is likely that this signalling cascade forms an important part of the mechanotransduction pathway that determines the response of chondrocytes to applied load.