sq-23377 and inositol-1-3-4-5-tetrakisphosphate

The endoplasmic reticulum (ER) is a dynamic organelle thought to consist of a single interconnected network of membranes. Using fluorescence recovery after photobleaching (FRAP) of HEK-293 cells dually transfected with soluble fluorescent proteins targeted to the ER (GFP) and mitochondria (DsRed), we have confirmed this continuity, which contrasts that of the mitochondria, which behave as a population of discrete organelles. The degree of ER integrity (interconnected versus fragmented) has been suggested to be regulated in some cells by inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P(4)). In HEK-293 and freshly isolated murine lacrimal acinar cells, we manipulated ER structure by disrupting cellular Ca(2+) homeostasis with the Ca(2+) ionophore ionomycin, and by permeabilisation of the plasma membrane, protocols known to cause ER fragmentation. However, we were subsequently unable to detect by FRAP any significant effect of Ins(1,3,4,5)P(4) on ER integrity.

Topics: Animals; Calcium Signaling; Cell Line; Cell Membrane; Endoplasmic Reticulum; Epithelial Cells; Fluorescence Recovery After Photobleaching; Green Fluorescent Proteins; Humans; Inositol Phosphates; Intracellular Membranes; Ionomycin; Ionophores; Lacrimal Apparatus; Mice; Mitochondria

All 9 racemic regioisomers (15 enantiomerically) of myo-inositol tetrakisphosphates (IP4s): DL-Ins(1,2,4,5)P4 [A], DL-Ins(1,2,4,6)P4 [B], Ins(1,2,3,5)P4 [C], Ins(1,3,4,6)P4 [D], Ins(2,4,5,6)P4 [E], DL-Ins(1,3,4,5)P4 [F], DL-Ins(1,2,5,6)P4 [G], DL-Ins(1,2,3,4)P4 [H] and DL-Ins(1,4,5,6)P4 [I] [Chung S-K., Chang Y-T. Synthesis of all possible regioisomers of myo-inositol tetrakisphosphate. J Chem Soc Chem Commun 1995; 11-13] were investigated for their ability to bind to the D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptor in bovine adrenal cortical membranes, and for their ability to mobilize 45Ca2+ from Ins(1,4,5)P3-sensitive Ca2+ stores in permeabilized Chinese hamster ovary (CHO) cells. DL-Ins(1,2,4,5)P4 (Ki = 11 nM) bound to Ins(1,4,5)P3 receptors with an affinity only 2-fold lower than Ins(1,4,5)P3 (Ki = 6 nM). Ins(1,2,3,5)P4, Ins(1,3,4,6)P4, Ins(2,4,5,6)P4, DL-Ins(1,3,4,5)P4, DL-Ins(1,2,3,4)P4 and DL-Ins(1,4,5,6)P4 bound with affinities of between 0.4-0.7 microM. DL-Ins(1,2,4,6)P4 and DL-Ins(1,2,5,6)P4 bound to the Ins(1,4,5)P3 receptor with low affinity (approximately 2-3 microM). All but one of the IP4s mediated release of 45Ca2+ from stores of permeabilized CHO cells with a similar rank order of potency as that for Ins(1,4,5)P3 receptor binding, being between 16-fold and 50-fold less potent at releasing 45Ca2+ compared with their apparent binding affinities to the Ins(1,4,5)P3 receptor. The notable exception was Ins(1,2,3,5)P4, which showed an approximately 200-fold lower potency compared with its affinity for the Ins(1,4,5)P3 receptor. Ins(1,2,3,5)P4 may be a useful lead compound for the rational design of novel synthetic Ins(1,4,5)P3 analogues possessing structure-activity profiles with relatively high binding affinity, but low intrinsic efficacy, and hence partial agonists and antagonists at the Ins(1,4,5)P3 receptor.

Topics: Animals; Calcium; Calcium Channels; Cattle; Cell Membrane Permeability; CHO Cells; Cricetinae; Inositol 1,4,5-Trisphosphate Receptors; Inositol Phosphates; Ionomycin; Ionophores; Isomerism; Models, Chemical; Receptors, Cytoplasmic and Nuclear

Ionomycin, a Ca2+ ionophore, stimulated phosphoinositide breakdown in rat brain cortical slices incubated in the presence of 1.2 mM Ca2+, but, unlike muscarinic cholinergic stimulation, it had little effect on inositol 1,3,4,5-tetrakisphosphate accumulation. However, at 2 min, the increase in inositol 1,4,5-trisphosphate due to 10 microM ionomycin was equivalent to that seen with 1 mM carbachol. Phorbol 12-myristate 13-acetate or high K+ (30 mM) increased inositol 1,4,5-trisphosphate, but not inositol 1,3,4,5-tetrakisphosphate accumulation. The stimulation of inositol 1,4,5-trisphosphate accumulation due to ionomycin, unlike that seen with carbachol, was abolished in buffer containing 0.2 mM Ca2+. The increase in inositol 1,3,4,5-tetrakisphosphate accumulation in brain slices due to 1 mM carbachol ranged from 55 to 68% of that for inositol 1,4,5-trisphosphate. Norepinephrine, NMDA, veratridine, and ouabain also increased inositol 1,4,5-trisphosphate, but had minimal effects on inositol 1,3,4,5-tetrakisphosphate accumulation. These results suggest that there is something unique about the stimulation of inositol 1,3,4,5-tetrakisphosphate accumulation by carbachol, which is also the only one of these agents that is able to activate phosphoinositidase C beta 1 in isolated rat brain membranes.

Topics: Animals; Atropine; Calcium; Carbachol; Cerebral Cortex; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Intracellular Membranes; Ionomycin; Male; Rats; Rats, Sprague-Dawley; Stimulation, Chemical; Tetradecanoylphorbol Acetate

Extracellular calcium concentrations markedly affect the pattern of proliferation and differentiation in cultured keratinocytes. When medium contains 0.1 mM calcium or above, the cells lose their proliferative ability, rapidly stratify, and terminally differentiate. Because 1,25(OH)2D3 (a modulator of Ca++ homeostasis) enhances the differentiation of keratinocytes, we investigated whether a link exists between 1,25(OH)2D3-induced release of inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) from PtdIns 4,5-P2 and intracellular calcium [Ca++]i release from keratinocytes. Specifically, primary culture of keratinocytes were loaded with fluorescence dye Fura-2AM (10 microM) and changes in fluorescence intensity were monitored at the excitation wavelengths of 340 and 380 nm and emission wavelength of 505 nm. Additions of two agonists, 1,25(OH)2D3 (1.2 x 10(-9) M) and 13-Cis retinoic acid (0.2 x 10(-9) M), to dye-loaded keratinocytes induced rapid release of [Ca++]i, respectively, followed by gradual return to the prestimulated state. Addition of Ins(1,4,5)P3 (10 microM) to saponin-treated (leaky) keratinocytes also resulted in a rapid release of [Ca++]i. In contrast, the addition of inositol-1,3,4,5-tetrakisphosphate Ins(1,3,4,5)P4 at similar concentrations exerted negligible effect. Taken together, these results support the view that 1,25(OH)2D3-induced [Ca++]i release in keratinocytes may be via the Ins(1,4,5)P3-induced early release of intracellular [Ca++]i. This may explain, at least in part, 1,25(OH)2D3-enhanced keratinocyte differentiation.

Topics: Animals; Animals, Newborn; Calcitriol; Calcium; Cell Differentiation; Cells, Cultured; Egtazic Acid; Fluorescent Dyes; Fura-2; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Ionomycin; Keratinocytes; Kinetics; Mice; Mice, Inbred BALB C; Spectrometry, Fluorescence; Tretinoin

Addition of bradykinin to mouse N1E-115 neuroblastoma cells evokes a rapid but transient rise in cytoplasmic free Ca2+ concentration ([Ca2+]i). The [Ca2+]i rise is accompanied by a transient membrane hyperpolarization, due to a several-fold increase in K+ conductance, followed by a prolonged depolarizing phase. Pretreatment of the cells with a Ca2+-ionophore abolishes the hormone-induced hyperpolarization but leaves the depolarizing phase intact. The transient hyperpolarization can be mimicked by iontophoretic injection of IP3(1,4,5) or Ca2+, but not by injection of IP3(1,3,4), IP4(1,3,4,5) or Mg2+ into the cells. Instead, IP3(1,3,4) evokes a small but significant membrane depolarization in about 50% of the cells tested. Microinjected IP4(1,3,4,5) has no detectable effect, nor has treatment of the cells with phorbol esters. These results suggest that, while IP3(1,4,5) triggers the release of stored Ca2+ to hyperpolarize the membrane, IP3(1,3,4) may initiate a membrane depolarization.

Topics: Animals; Bradykinin; Calcium; Cell Line; Electrophysiology; Ethers; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Ionomycin; Membrane Potentials; Microinjections; Neuroblastoma; Sugar Phosphates

We have earlier reported that Inositol (1,3,4,5)tetrakisphosphate microinjection will activate eggs of the sea urchin Lytechinus variegatus provided that it is co-injected with inositol (2,4,5)trisphosphate (Irvine and Moor, Biochem. J. 240, 917-920, 1986). Here we extend these observations to show that inositol (1,3,4,5,6)pentakisphosphate is a partial agonist in this assay and the requirement for the presence of inositol (2,4,5)trisphosphate cannot be bypassed by raised, but sub-threshold, Ca2+ concentrations. A mechanism for the proposed stimulation of Ca2+ entry into the cell requiring both inositol tris- and tetrakisphosphates is presented.

Topics: Animals; Calcium; Ethers; Female; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Ionomycin; Oocytes; Sea Urchins; Sugar Phosphates