Light induces a rapid and transient increase in inositol-trisphosphate in toad rod outer segments

The sub-second time course of changes in the content of [3H]inositol-1,4,5-trisphosphate was determined in rod outer segments from very rapidly frozen Bufo retinas that had been incubated with [3H]inositol. Rod outer segments were cut off frozen specimens with a cryostat microtome and the water soluble extracts were analyzed. The content of [3H]inositol-1,4,5-trisphosphate rose after approximately 250 msec of bright illumination, but returned to the unstimulated level after 1 sec, whether the stimulus remained on or not. That is, there was rapid but transient change in the content of [3H]inositol-1,4,5-trisphosphate after the onset of stimulation.     

Inositol-1,4,5-trisphosphate releases calcium from skinned cultured smooth muscle cells

We examined the effects of inositol-1,4,5-trisphosphate on 45Ca uptake and 45Ca efflux in the saponin skinned primary cultured rat aortic smooth muscle cells. 10 microM inositol-1,4,5-trisphosphate induced a rapid (half time less than 10 sec) and large quantity of Ca release in both 45Ca uptake and 45Ca efflux in the skinned cells preloaded with 1 microM free Ca. Dose response curves showed that 100 microM inositol-1,4,5-trisphosphate produced a maximal Ca release of 97.3% of the MgATP dependent 45Ca uptake or 289 mumoles/liter cells, which was much greater than the maximal caffeine induced Ca release and would be sufficient to produce maximal tension.     

Release of Ca2+ from plant hypocotyl microsomes by inositol-1,4,5-trisphosphate

The effect of inositol-1,4,5-trisphosphate on Ca2+ release from microsomes isolated from dark-grown zucchini (Cucurbita pepo L.) hypocotyls was studied. Up to 30% of the Ca2+ taken up by the microsomes in the presence of 2mM ATP, was released by mumolar concentrations of inositol-1,4, 5-trisphosphate. This release was very rapid (less than 0.5 min) and was followed by a slower re-uptake of Ca2+. The microsomal levels of Ca2+ previously attained were not re-established within 5 min. External concentration of free Ca2+ was maintained in the 10(-8)M region during the release. This is the first time that inositol-1,4,5-trisphosphate has been shown to have a regulatory effect on Ca2+ in plant membrane fractions. Phosphoinositides may be important in signal transduction in plant cells, by altering the cytoplasmic Ca2+ activity, a function already known in animal cells.     

Muscarinic receptor-induced phosphoinositide hydrolysis at resting cytosolic Ca2+ concentration in PC12 cells

In PC12 cells, cultured in the presence of nerve growth factor to increase their complement of muscarinic receptors, treatment with carbachol induces muscarinic receptor-dependent rises in free cytosolic Ca2+ as well as hydrolysis of membrane phosphoinositides. Experiments were carried out to clarify the relationship between these two receptor-triggered events. In particular, since inositol-1,4,5-trisphosphate (the hydrophilic metabolite produced by the hydrolysis of phosphatidylinositol-4,5-bisphosphate) is believed to mediate intracellularly the release of Ca2+ from nonmitochondrial store(s), it was important to establish whether it can be generated at resting cytoplasmic concentration of Ca2+ (approximately 0.1 microM). Cells incubated in Ca2+-free medium were depleted of their cytoplasmic Ca2+ stores by pretreatment with ionomycin. When these cells were then treated with carbachol, their cytosolic concentration of Ca2+ remained at the resting level, whereas inositol-1,4,5-trisphosphate generation was still markedly stimulated. Our results demonstrate that an increase in the concentration of cytosolic Ca2+ is not a necessary intermediate between receptor activation and phosphoinositide hydrolysis, and therefore support the second-messenger role of inositol-1,4,5-trisphosphate.     

A possible role for glucose metabolites in the regulation of inositol-1,4,5-trisphosphate 5-phosphomonoesterase activity in pancreatic islets

Rat pancreatic islets demonstrate inositol-1,4,5-trisphosphate 5-phosphomonoesterase activity which is 3 times higher than that in the exocrine pancreas. This enzyme has several features in common with the erythrocyte and hepatocyte enzymes: it is located primarily in the plasma membrane, it has a similar Km for inositol trisphosphate (IP3) (16 microM), and it requires Mg2+. The activity of the islet enzyme is inhibited by several diphosphorylated glucose metabolites: 2,3-bisphosphoglycerate, fructose 1,6-bisphosphate, fructose 2,6-bisphosphate, and glucose 1,6-bisphosphate. Monophosphorylated and unphosphorylated metabolites have little or no effect on its activity. Several reports show that stimulation of islets with glucose raises the concentrations of various glucose metabolites including fructose 1,6-bisphosphate, glucose 1,6-bisphosphate, and 2,3-bisphosphoglycerate to concentrations that are in the range that inhibit the islet inositol-1,4,5-trisphosphate 5-phosphomonoesterase. Other reports show that IP3 mobilizes calcium when added to permeabilized insulin-secreting cells. It is possible that the increase in cytosolic calcium known to occur during glucose-induced insulin secretion may be sustained in part by higher IP3 levels resulting from the inhibition of inositol-1,4,5-trisphosphate 5-phosphomonoesterase by some of the diphosphorylated glucose metabolites.     

Different effects of platelet-derived growth factor isoforms on rat vascular smooth muscle cells

The response of rat aortic smooth muscle cells to all three isoforms of platelet-derived growth factor (PDGF) was studied. 5,000 binding sites/cell were estimated for rPDGF-AA (Kd 0.22 nM), 45,000 for rPDGF-AB and (Kd 0.4 nM), and 31,000 for rPDGF-BB (Kd 0.29 nM). rPDGF-AB and -BB stimulated effectively [3H]thymidine incorporation, inositol 1,4,5-trisphosphate release, diacylglycerol productions, [Ca2+]i increase, and pHi changes at concentration in the range from 3 to 10 ng/ml. The extent of DNA synthesis stimulated by rPDGF-AA varied considerably, and in all cases higher concentrations than 10 ng/ml were required. rPDGF-AA did not stimulate inositol-1,4,5-trisphosphate release, [Ca2+]i increase or pHi changes but induced the production of diacylglycerol, although with a different kinetic compared with that observed with rPDGF-AB or -BB. Apparently rPDGF-AA acts via a different mechanism, generating diacylglycerol without the release of inositol-1,4,5-trisphosphate.     

Evidence for a lack of inositol--(1,4,5)trisphosphate kinase activity in norepinephrine-perfused rat hearts

The products of the phosphatidylinositol turnover pathway in norepinephrine-perfused hearts have been examined using high performance liquid chromatography. Inositol-1 phosphate, inositol-(1,4)bisphosphate and inositol-(1,4,5)-trisphosphate were all increased in response to norepinephrine stimulation. However, at perfusion times from 5 sec to 20 min there was no appearance of inositol-(1,3,4,5)tetrakisphosphate or inositol-(1,3,4)trisphosphate. This suggests the absence of the inositol-(1,4,5)trisphosphate phosphorylation/dephosphorylation pathway in heart and demonstrates that this secondary pathway is not essential to the functioning of the phosphatidylinositol turnover cycle.     

Inositol-1,4,5-trisphosphate and non-hydrolysable GTP analogue induced calcium release from intracellular stores of the Helix pomatia neurons

1. Cytoplasmic Ca2+ concentration ICaIin and membrane potential of single Helix pomatia neurons was studied by Fura-2 fluorescence measurement and conventional current clamp methods. 2. Intracellular injection of inositol-1,4,5-trisphosphate (IP3) and nonhydrolysable GTP analogue (Gpp/NH/p) led to a rise of ICaIin; in contrast, GTP injection did not cause significant ICaIin changes. 3. We suggest that both IP3 and Gpp/NH/p directly activated Ca release from intracellular stores.     

The calcium-mobilizing effect of inositol-1,4,5-triphosphate in rod outer segments and disks]

The effect of inositol-1,4,5-trisphosphate (IP3) on the release of calcium ions from retinal rod discs was studied. It was shown that the release of Ca2+ from discs is an electroneutral process. The intradiscal calcium concentration during the release of the ion from the organelle decreases by 1 mM. It was found that the IP3-dependent release of Ca2+ ions from discs is activated by guanosine triphosphate and beta gamma-transducin. The increase in calcium concentration in the medium also activates the IP3-dependent release of Ca2+ ions from discs, which probably is due to the stimulation of phospholipase C. It is suggested that the functional role of the release of ions in related not to phototransduction but to slow regulatory and adaptation processes in the photoreceptor cell.     

Targeted Activation of Conventional and Novel Protein Kinases C through Differential Translocation Patterns

Activation of the two ubiquitous families of protein kinases, protein kinase A (PKA) and protein kinase C (PKC), is thought to be independently coupled to stimulation of Gα_s and Gα_q, respectively. Live-cell confocal imaging of protein kinase C fluorescent protein fusion constructs revealed that simultaneous activation of Gα_s and Gα_q resulted in a differential translocation of the conventional PKCα to the plasma membrane while the novel PKCδ was recruited to the membrane of the endoplasmic reticulum (ER). We demonstrate that the PKCδ translocation was driven by a novel Gα_s-cyclic AMP-EPAC-RAP-PLCε pathway resulting in specific diacylglycerol production at the membrane of the ER. Membrane-specific phosphorylation sensors revealed that directed translocation resulted in phosphorylation activity confined to the target membrane. Specific stimulation of PKCδ caused phosphorylation of the inositol-1,4,5-trisphosphate receptor and dampening of global Ca²⁺ signaling revealed by graded flash photolysis of caged inositol-1,4,5-trisphosphate. Our data demonstrate a novel signaling pathway enabling differential decoding of incoming stimuli into PKC isoform-specific membrane targeting, significantly enhancing the versatility of cyclic AMP signaling, thus demonstrating the possible interconnection between the PKA and PKC pathways traditionally treated independently. We thus provide novel and elementary understanding and insights into intracellular signaling events.     

IP3-independent signalling of OX1 orexin/hypocretin receptors to Ca2+ influx and ERK

OX1 orexin receptors (OX1R) have been shown to activate receptor-operated Ca2+ influx pathways as their primary signalling pathway; however, investigations are hampered by the fact that orexin receptors also couple to phospholipase C, and therewith inositol-1,4,5-trisphosphate (IP3)-dependent Ca2+ release. We have here devised a method to block the latter signalling in order to focus on the mechanism of Ca2+ influx activation by OX1R in recombinant systems. Transient expression of the IP3-metabolising enzymes IP3-3-kinase-A (inositol-1,4,5-trisphosphate-->inositol-1,3,4,5-tetrakisphosphate) and type I IP3-5-phosphatase (inositol-1,4,5-trisphosphate-->inositol-1,4-bisphosphate) almost completely attenuated the OX1R-stimulated IP3 elevation and Ca2+ release from intracellular stores. Upon attenuation of the IP3-dependent signalling, the receptor-operated Ca2+ influx pathway became the only source for Ca2+ elevation, enabling mechanistic studies on the receptor-channel coupling. Attenuation of the IP3 elevation did not affect the OX1R-mediated ERK (extracellular signal-regulated kinase) activation in CHO cells, which supports our previous finding of the major importance of receptor-operated Ca2+ influx for this response.     

Ca influx evoked by inositol-3,4,5,6-tetrakisphosphate in ras-transformed NIH/3T3 fibroblasts

Infusion of inositol-3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P₄) from the patch pipette into the cytoplasm, produced a biphasic intracellular free Ca²⁺ concentration ([Ca²⁺]_i) increase in ras-transformed NIH/3T3 (DT) cells. The Ins(3,4,5,6)P₄-induced increase in DT cells depended upon extracellular Ca²⁺ and was enhanced by membrane hyperpolarization. Identical [Ca²⁺]_i increases were observed with intracellular application of inositol-1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P₄) and inositol-1,3,4,6-tetrakisphosphate but not with inositol-1,2,4,5-tetrakisphosphate, inositol-1,4,5-trisphosphate or inositol-1,3,4,5,6-pentakisphosphate. Stimulation of DT cells with bradykinin increased the levels of Ins(3,4,5,6)P₄ and Ins(1,3,4,5)P₄. These results suggest that Ins(3,4,5,6)P₄ may serve as a second messenger for continuous Ca²⁺ influx along with other tetrakisphosphates downstream from bradykinin receptors in DT cells.     

Dopamine Receptor Stimulation Does Not Affect Phosphoinositide Hydrolysis in Slices of Rat Striatum

The effect of dopamine receptor stimulation on the accumulation of labelled inositol phosphates in rat striatal slices under basal and stimulated conditions was examined following preincubation with [3H]inositol. Incubation of striatal slices with the selective D-1 agonist SKF 38393 or the selective D-2 agonist LY 171555 for 5 or 30 min did not affect the basal accumulation of labelled inositol mono-, bis-, tris-, and tetrakisphosphate. Resolution by HPLC of inositol trisphosphate into inositol-1,3,4-tris-phosphate and inositol-1,4,5-trisphosphate isomers revealed that under basal conditions dopamine did not influence the accumulation of inositol-1,4,5-trisphosphate. Depolarisation evoked by KCl, or addition of the muscarinic receptor agonist carbachol, produced a marked increase in the accumulation of labelled inositol phosphates in both the presence and absence of lithium. Addition of dopamine did not reduce the ability of KCl or carbachol to increase inositol phospholipid hydrolysis. In the presence of lithium, dopamine (100 microM) enhanced KCl-stimulated inositol phospholipid hydrolysis, but this effect appears to be mediated by alpha 1 adrenoceptors because it was blocked by prazosin. SKF 38393 (10 microM) or LY 171555 (10 microM) also did not affect carbachol-stimulated inositol phospholipid hydrolysis. These data, in contrast to recent reports, suggest that striatal dopamine receptors do not appear to be linked to inositol phospholipid hydrolysis.     

Inositol trisphosphate-induced hyperpolarization in rat dorsal root ganglion neurons.

Inositol 1,4,5-trisphosphate (1,4,5-InsP3) was perfused into rat dorsal root ganglion (DRG) neurons by whole-cell patch-clamp electrodes, while measuring the membrane potential. This operation evoked a transient (2-3 min) membrane hyperpolarization of about -15 mV (from -42 mV) followed by a depolarization. The membrane hyperpolarization was abolished when 30 mM EGTA was perfused together with 1,4,5-InsP3 or when 0.2 mM quinine was added to the bath solution. The hyperpolarizing response was enhanced when a low-Ca2+ EGTA-free intracellular solution was used. Two InsP2 isomers induced a different response. Our results suggest that the hyperpolarization is due to 1,4,5-InsP3-induced Ca2+ release which may trigger Ca-sensitive K+ channels to open. Present results show that cultured DRG neurons are able to respond to 1,4,5-InsP3 perfusion in the whole-cell configuration.     

20 years of Ins(1,4,5)P3, and 40 years before

This year marks the 20th birthday of the discovery of inositol-1,4,5-trisphosphate as a second messenger. The background to this discovery is a complex story that goes back more than 50 years and involves a large cast of characters, both chemical and human.     

Effects of N-n-butyl haloperidol iodide on L-type calcium channels and intracellular free calcium in rat ventricular myocytes.

The ability of N-n-butyl haloperidol iodide (F2) to cause vasodilation, and thereby produce a cardioprotective effect, has been well documented. The aim of this study was to investigate whether F2 might act as a Ca2+ antagonist. Myocytes were obtained from rat heart, and the whole-cell patch-clamp technique was used to record Ca2+ current. Laser scanning confocal microscopy was used to measure intracellular free calcium ([Ca2+]i). The results obtained from this study demonstrate that F2 reduced calcium current (ICa) in a concentration-dependent manner with an IC50 of 1.19 micromol/L, upshifted the current-voltage curve of ICa, shifted the inactivation kinetics of ICa leftward, and slowed down the recovery of ICa from inactivation. F2 decreased the fluorescent intensity of [Ca2+]i elevation induced by KCl with an IC50 of 1.61 micromol/L, and had no effects on the intracellular calcium release induced by caffeine and inositol-1,4,5-trisphosphate. These findings indicate that F2 may act as a calcium antagonist, which could account for its cardiovascular benefits.     

Stimulations of arachidonate release and inositol-1,4,5-triphosphate formation are mediated by distinct G-proteins in human platelets

Addition of fluoroaluminate to human platelet suspension stimulated thromboxane synthesis and inositol-1,4,5-triphosphate formation in a time and dose dependent manner. Neomycin inhibited markedly fluoroaluminate induced inositol-1,4,5-triphosphate formation without significantly affecting thromboxane synthesis. Preincubation of platelets with PGE1, also inhibited significantly inositol-1,4,5-triphosphate formation with modest reduction of thromboxane synthesis. On the contrary, pretreatment of platelets with pertussis toxin inhibited fluoroaluminate stimulated thromboxane synthesis without affecting inositol-1,4,5-triphosphate formation. Similarly, preincubation of platelets with phorbol ester, PMA, inhibited markedly thromboxane synthesis with modest reduction of inositol-1,4,5-triphosphate formation. These results indicate that inositol-1,4,5-triphosphate formation and arachidonate release and thromboxane synthesis are controlled separately and are mediated by different G-proteins which are coupled to phospholipase C and phospholipase A2 respectively in platelets.     

Fibroblast growth factor-2 stimulates phospholipase Cbeta in adult cardiomyocytes.

Although fibroblast growth factor-2 (FGF-2) plays an important role in cardioprotection and growth, little is known about the signals triggered by it in the adult heart. We therefore examined FGF-2-induced effects on phosphoinositide-specific phospholipase C (PI-PLC) isozymes, which produce second messengers linked to the inotropic and hypertrophic response of the myocardium. FGF-2, administered by retrograde perfusion to the isolated heart, induced an increase in inositol-1,4,5-trisphosphate levels in the cytosol, as well as an increase in total PI-PLC activity associated with sarcolemmal and cytosolic fractions. Furthermore FGF-2 induced a time-dependent elevation in cardiomyocyte membrane-associated PLC gamma1 and PLC beta1 activities, assayed in immunoprecipitated fractions, and moreover, increased the membrane levels of PLC beta1 and PLC beta3. Activation of PLC beta is suggestive of FGF-2-induced cross-talk between FGF-receptor tyrosine kinase and G-protein-coupled signaling in adult cardiomyocytes and underscores the importance of FGF-2 in cardiac physiology.     

Phosphatidylinositol 4,5-bisphosphate specific phospholipase C inPharbitis nil membranes

Phosphatidylinositol 4,5-bisphosphate specific phospholipase C has been detected in a membrane preparation fromPharbitis nil cotyledons. The enzyme has a pH optimum of 6.8 and activated by calcium ions, deoxycholate, phosphatidylinositol and phosphatidylethanolamine. The enzyme is inhibited to varying degrees by Tween 20, Triton XI00, zinc, copper, cobalt and manganese ions and phosphatidylserine. G-protein activators do not affect the activity ofPharbitis nil phospholipase C. Analysis of the products of the reaction by HPLC shows inositol 1,4,5-trisphosphate from phospholipase C and inositol bisphosphate from inositol-1 and -5 phosphatase activity.     

Back in the water: the return of the inositol phosphates

Following the discovery of inositol-1,4,5-trisphosphate as a second messenger, many other inositol phosphates were discovered in quick succession, with some understanding of their synthesis pathways and a few guesses at their possible functions. But then it all seemed to go comparatively quiet, with an explosion of interest in the inositol lipids. Now the water-soluble phase is once again becoming a focus of interest. Old and new data point to a new vista of inositol phosphates, with functions in many diverse aspects of cell biology, such as ion-channel physiology, membrane dynamics and nuclear signalling.