Alpha 1-adrenergic activation of brown adipocytes leads to an increased formation of inositol polyphosphates

alpha 1-Adrenergic activation of isolated brown adipocytes causes a rapid mobilization of intracellular Ca2+. The cells also respond with an increased turnover of inositol lipids. The present work demonstrates that alpha 1-adrenergic stimulation of brown adipocytes results in phospholipase C-mediated breakdown of phosphatidylinositol bisphosphate to form inositol trisphosphate. The rate of appearance of inositol trisphosphate is sufficiently rapid for it to mediate or contribute to Ca2+ mobilization in these cells.     

Inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate formation in Ca2+-mobilizing-hormone-activated cells.

The inositol trisphosphate liberated on stimulation of guinea-pig hepatocytes, pancreatic acinar cells and dimethyl sulphoxide-differentiated human myelomonocytic HL-60 leukaemia cells is composed of two isomers, the 1,4,5-trisphosphate and the 1,3,4-trisphosphate. Inositol 1,4,5-trisphosphate was released rapidly, with no measurable latency on hormone stimulation, and, consistent with its proposed role as an intracellular messenger for Ca2+ mobilization, there was good temporal correlation between its formation and Ca2+-mediated events in these tissues. There was a definite latency before an increase in the formation of inositol 1,3,4-trisphosphate could be detected. In all of these tissues, however, it formed a substantial proportion of the total inositol trisphosphate by 1 min of stimulation. In guinea-pig hepatocytes, where inositol trisphosphate increases for at least 30 min after hormone application, inositol 1,3,4-trisphosphate made up about 90% of the total inositol trisphosphate by 5-10 min. In pancreatic acinar cells, pretreatment with 20 mM-Li+ caused an increase in hormone-induced inositol trisphosphate accumulation. This increase was accounted for by a rise in inositol 1,3,4-trisphosphate; inositol 1,4,5-trisphosphate was unaffected. This finding is consistent with the observation that Li+ has no effect on Ca2+-mediated responses in these cells. The role, if any, of inositol 1,3,4-trisphosphate in cellular function is unknown.     

IP3 3-kinase opposes NGF driven neurite outgrowth

The inositol (1,4,5) trisphosphate 3-kinases comprise a family of enzymes (A, B, and C) that phosphorylate the calcium mobilising molecule inositol (1,4,5) trisphosphate (IP(3)) to generate inositol (1,3,4,5) tetrakisphosphate. This molecule can function as a second messenger, but its roles are not completely understood. The A isoform of inositol (1,4,5) trisphosphate 3-kinase localises to filamentous actin within dendritic spines in the hippocampus and is implicated in the regulation of spine morphology and long term potentiation, however the mechanisms through which it signals in neuronal cells are not completely understood. We have used NGF driven neurite outgrowth from PC12 cells as a platform to examine the impact of signaling via inositol (1,4,5) trisphosphate 3-kinase activity in a neuronal cell. We have found that the catalytic activity of the enzyme opposes neurite outgrowth, whilst pharmacological inhibition of inositol (1,4,5) trisphosphate 3-kinase leads to a significant increase in neurite outgrowth, and we show that the reduction in neurite outgrowth in response to inositol (1,4,5) trisphosphate 3-kinase activity correlates with reduced ERK activity as determined by western blotting using phosphorylation-specific antibodies. Our findings suggest a novel neuronal signaling pathway linking metabolism of IP(3) to signaling via ERK.     

Properties of receptor-controlled inositol trisphosphate formation in parotid acinar cells.

Activation of muscarinic receptors in rat parotid cells results in breakdown of polyphosphoinositides liberating inositol phosphates, including inositol trisphosphate. Formation of inositol trisphosphate appears independent of agonist-induced Ca2+ mobilization, since neither formation nor degradation of inositol trisphosphate are appreciably altered in low-calcium media, and elevation of cytosolic Ca2+ with a calcium ionophore does not cause an increase in cellular inositol trisphosphate. Further, activation of substance P receptors and alpha 1-adrenoreceptors, but not beta-adrenoreceptors, increases inositol trisphosphate formation. The dose-response curve for methacholine activation of inositol trisphosphate formation more closely approximates the curve for receptor occupancy than for Ca2+-activated K+ release. These results are all consistent with the suggestion that inositol trisphosphate could function as a second messenger linking receptor occupation to cellular Ca2+ mobilization.     

Domains for G-protein coupling in angiotensin II receptor type I: studies by site-directed mutagenesis.

To delineate domains essential for G-protein coupling in angiotensin II type 1 receptor (AT1), we mutated the receptor cDNA in the putative cytosolic regions and determined consequent changes in the effect of GTP analogs on angiotensin II (Ang II) binding and in inositol trisphosphate production in response to Ang II. Polar residues in targeted areas were replaced by small neutral residues. Mutations in the second cytosolic loop, carboxy terminal region of the third cytosolic loop or deletional mutation in the carboxyl terminal tail simultaneously abolished both the GTP-induced shift to the low affinity form and Ang II-induced stimulation of inositol trisphosphate production. These results suggest that polar residues in the second cytosolic loop, the carboxy terminal region of the third cytosolic loop, and the carboxy terminal cytosolic tail are important for G-protein coupling of AT1 receptor.     

Intracellular Ca(2+) release channels in evolution

Intracellular Ca(2+)-release channels (ICRCs) form a superfamily of genes that encompasses two distinct subfamilies: the inositol trisphosphate receptor and the ryanodine receptor genes, which encode the largest ion channels known today. During evolution from nematodes to man, mechanisms of gene duplication and divergence have increased the number of known ICRC genes, which have been gradually co-opted to contribute to the increasing complexity of intracellular Ca(2+) signalling required for regulation of specialised eukaryotic cell activities.     

Decreased formation of inositol trisphosphate in Madin-Darby canine kidney cells under conditions of beta-glucosidase inhibition.

Recent work has demonstrated the enhancement of hormone-stimulated inositol trisphosphate formation in renal epithelial cells under conditions of glucosylceramide depletion. The role of glucosylceramide metabolism was explored further by exposing Madin-Darby canine kidney (MDCK) cells to the beta-glucosidase inhibitor conduritol B epoxide, which produced time-dependent and concentration-dependent increases in glucosylceramide levels and decreased bradykinin-stimulated inositol trisphosphate formation from isolated MDCK cell membranes. These data provide further support for an association between glucosylceramide levels and hormone-stimulated inositol trisphosphate formation.     

Ca2+ release by inositol trisphosphate from Ca2+-transporting microsomes derived from uterine sarcoplasmic reticulum

Microsomes derived from pregnant uterine sarcoplasmic reticulum, isolated by differential and sucrose density gradient centrifugation, accumulates Ca2+ in the presence of ATP. Inositol trisphosphate caused release of this Ca2+, in a dose dependent manner. 40% of the Ca2+ that can be released by the ionophore A23187 was released by 5 microM inositol trisphosphate. Removal of Mg by EDTA prior to addition of inositol trisphosphate did not change the course of Ca2+ release. These results indicate that by mobilizing intracellular Ca2+, inositol trisphosphate may be the link between hormonal stimuli and smooth muscle contraction.     

Inositol trisphosphate stimulates the release of calcium from intact vacuoles isolated from Acer cells

On addition of inositol trisphosphate, intact vacuoles isolated from Acer pseudoplatanus cell suspension cultures release part of their calcium content. The process was specific, dose-dependent (IC₅₀ = 0.2μM) and was inhibited by an intracellular calcium antagonist. The calcium efflux elicited by inositol trisphosphate increased with the age of the cell suspension cultures, the maximum effect being obtained when the cultures reached the stationary phase. It is suggested that vacuoles play a role as an endocellular calcium store that is responsive to inositol trisphosphate in plants.     

Hemoglobin causes release of inositol trisphosphate from vascular smooth muscle

To test the hypothesis that oxyhemoglobin causes contraction of vascular smooth muscle by production of inositol 1,4,5-trisphosphate which results in a release of intracellular calcium, smooth muscle cells were exposed to oxyhemoglobin and inositol trisphosphate was measured. Oxyhemoglobin, but not methemoglobin which has much less contractile action, stimulated inositol trisphosphate production. The time course was consistent with an early role for this compound in the contraction produced by hemoglobin. The increase in production of inositol trisphosphate was inhibited by pertussis toxin and also by neomycin, an inhibitor of phospholipase C, although the actions of the latter compound cannot be attributed only to an inhibition of the enzyme responsible for the production of inositol trisphosphate.     

Metabolism of inositol phosphates in parotid cells: implications for the pathway of the phosphoinositide effect and for the possible messenger role of inositol trisphosphate

Rat parotid acinar cells were used to investigate the time course of formation and breakdown of inositol phosphates in response to receptor-active agents. In cells preincubated with [3H]inositol and in the presence of 10 mM LiCl (which blocks hydrolysis of inositol phosphate), methacholine (10(-4)M) caused a substantial increase in cellular content of [3H]inositol phosphate, [3H]inositol bisphosphate and [3H]inositol trisphosphate. Subsequent addition of atropine (10(-4) M) caused breakdown of [3H]inositol trisphosphate and [3H]inositol bisphosphate and little change in accumulated [3H]inositol phosphate. The data could be fit to a model whereby inositol trisphosphate and inositol bisphosphate are formed from phosphodiesteratic breakdown of phosphatidylinositol bisphosphate and phosphatidylinositol phosphate respectively, and inositol phosphate is formed from hydrolysis of inositol bisphosphate rather than from phosphatidyl-inositol. Consistent with this model was the finding that [3H]inositol trisphosphate and [3H]inositol bisphosphate levels were substantially increased in 5 sec while an increase in [3H]inositol phosphate was barely detectable at 60 sec. These results indicate that in the parotid gland the phosphoinositide cycle is activated primarily by phosphodiesteratic breakdown of the polyphosphoinositides rather than phosphatidyl-inositol. Also, the results show that formation of inositol trisphosphate is probably sufficiently rapid for it to act as a second messenger signalling internal Ca2+ release in this tissue.     

Inositol phospholipid turnover in platelets of schizophrenic patients.

Abnormalities in blood cell membrane phospholipid composition and metabolism from schizophrenic patients have been reported by many groups of investigators. Among membrane phospholipids, inositol phospholipids are of special importance as they are involved in transduction system that generates second messengers such as inositol trisphosphate and diacylglycerol. Our studies on platelet inositol phospholipid turnover suggest a significant increase in platelet phosphatidylinositol 4,5-bisphosphate levels, an increased production of inositol trisphosphate in neuroleptic-treated and neuroleptic-free schizophrenic patients platelets and a reduced calcium release by thrombin in neuroleptic-treated schizophrenic patients platelets. The enhanced production of inositol trisphosphate may be due to an increase in its precursor phosphatidylinositol 4,5-bisphosphate with an associated desensitisation of the intracellular inositol trisphosphate receptor by neuroleptics, which may explain the diminished calcium response to thrombin in schizophrenic patients platelets.     

Inositol trisphosphates in carbachol-stimulated rat parotid glands.

Carbachol stimulation of rat parotid gland fragments prelabelled with myo-[3H]-inositol results in a large accumulation after 15 min of [3H]inositol trisphosphate. Only some of this is the D-1,4,5 isomer which would be expected to be derived from the known phosphatidylinositol bisphosphate. The predominant inositol trisphosphate is not susceptible to hydrolysis by human erythrocyte membranes. It yields altritol after periodate treatment followed by reduction and dephosphorylation, and, from partial dephosphorylation experiments, does not have a phosphate in the 2 position; the most likely structure of this inositol trisphosphate is therefore (D/L)-myo-inositol 1,3,4-trisphosphate. The possible origin and significance of this compound are discussed.     

Intracellular Ca release channels in evolution

Intracellular Ca²⁺-release channels (ICRCs) form a superfamily of genes that encompasses two distinct subfamilies: the inositol trisphosphate receptor and the ryanodine receptor genes, which encode the largest ion channels known today. During evolution from nematodes to man, mechanisms of gene duplication and divergence have increased the number of known ICRC genes, which have been gradually co-opted to contribute to the increasing complexity of intracellular Ca²⁺ signalling required for regulation of specialised eukaryotic cell activities.     

The conserved Arg241‐Glu439 salt bridge determines flexibility of the inositol 1,4,5‐trisphosphate receptor binding core in the ligand‐free state

Inositol 1,4,5‐trisphosphate receptor (InsP₃R) is an intracellular Ca²⁺‐release channel activated by binding of inositol 1,4,5‐trisphosphate (InsP₃) to the InsP₃ binding core (IBC). Structural change in the IBC upon InsP₃ binding is the key process in channel pore opening. In this study, we performed molecular dynamics (MD) simulations of the InsP₃‐free form of the IBC, starting with removal of InsP₃ from the InsP₃‐bound crystal structure, and obtained the structural ensemble of the InsP₃‐free form of the IBC. The simulation revealed that the two domains of the IBC largely fluctuate around the average structure with the hinge angle opened 17° more than in the InsP₃‐bound form, and the twist angle rotated by 45°, forming interdomain contacts that are different from those in the bound form. The InsP₃ binding loop was disordered. The InsP₃‐free form thus obtained was reproduced four times in simulations started from a fully extended configuration of the two domains. Simulations beginning with the fully extended form indicated that formation of a salt bridge between Arg241 and Glu439 is crucial for stabilizing the closed form of the two domains. Mutation of Arg241 to Gln prevented formation of the compact structure by the two domains, but the fully flexible domain arrangement was maintained. Thus, the Arg241‐Glu439 salt bridge determines the flexibility of the InsP₃‐free form of the IBC.Proteins 2013; 81:1699–1708. © 2013 Wiley Periodicals, Inc.     

Is vacuole the richest store of IP3-mobilizable calcium in plant cells?

Inositol trisphosphate is known to mobilize calcium from internal stores in plant cells. However, with the exception of the vacuole, the largest plant cell compartment, organelles responsive to inositol trisphosphate have not been extensively identified. In this way, we have separated membrane vesicles from the same carrot microsomal fraction and identified them, both by marker enzyme activities and electron microscopy. These correspond to pure plasma membrane, pure tonoplast and mixed mitochondria, endoplasmic reticulum, Golgi membrane fractions. All the fractions accumulated calcium in a ATP-dependent manner and were tightly sealed. Inositol trisphosphate-dependent calcium releases were accurately measured only in fractions corresponding functionally and structurally to tonoplast, the vacuolar membrane. The process was dose-dependent and fairly specific for inositol trisphosphate. While highly significant, approximately 40% of the mobile calcium only may be released from tonoplast vesicles by inositol trisphosphate which remained basically intact during the release experiments. From these results it is concluded that the vacuole is the richest store of calcium directly mobilizable by inositol trisphosphate in plant cells, but inositol trisphosphate is not able to release the overall mobile vacuolar calcium.     

Alpha 1-adrenergic inositol trisphosphate production in brown adipocytes is Na+ dependent

In order to investigate the ionic requirements for inositol trisphosphate production, brown adipocytes were prelabelled with myo-[3H]inositol and the formation of inositol trisphosphates and inositol bisphosphates as a consequence of alpha 1-adrenergic stimulation was monitored. Omission of Ca2+ from the incubation medium diminished the norepinephrine-induced increase in inositol trisphosphate levels, but it would seem that this reduction can be fully accounted for by a decreased level of the 'inactive' isomer inositol 1,3,4-trisphosphate. Omission of Na+ fully abolished the norepinephrine-induced inositol trisphosphate response. However, it was observed that the presence of Li+ in the incubation medium could fully reconstitute the ability of the cells to yield the early response of inositol trisphosphate production; Li+ could, however, not substitute for Na+ in the entire alpha 1-adrenergic cellular pathway. It was concluded that the Na+-dependent step is found in the coupling mechanism between the alpha 1-receptor and the activation of the phosphodiesterase responsible for inositol trisphosphate production. Thus, all events in the alpha 1-adrenergic pathway which are consequences of IP3 production should appear to be Na+-dependent in these cells.     

Formation of inositol pentakisphosphate by ovarian follicles of Xenopus laevis from metabolism of inositol (1,4,5)trisphosphate and inositol (1,3,4,5)tetrakisphosphate and from receptor activation

Small amounts of a higher inositol phosphate with chromatographic properties of [3H]inositol (1,3,4,5,6)pentakisphosphate were formed from [3H]inositol (1,4,5)trisphosphate added to homogenates of ovarian follicles of Xenopus laevis, and from [3H]inositol (1,3,4,5)tetrakisphosphate after injection into follicular oocytes. Other intermediate forms of inositol tetrakisphosphate were not detectable. [3H]inositol (1,3,4,5,6)pentakisphosphate prepared from chicken erythrocytes was metabolized in homogenates to an inositol tetrakisphosphate eluting later than the (1,3,4,5) isomer. Activation of receptors in ovarian follicles of Xenopus laevis with acetylcholine or stimulation with injected GTP gamma S caused formation not only of inositol trisphosphate and its expected metabolites but also of small amounts of inositol pentakisphosphate. These results suggest that the latter may be formed from metabolites of inositol (1,4,5)trisphosphate in this tissue during receptor activation.     

The NK-1 Receptor and a Calcium-Phospholipid Pathway: Inositol Trisphosphate Production and Calcium Movements Induced by Selective Agonists of Neurokinin Receptors in Rat Parotid Glands

In the rat parotid gland, substance P has been shown to induce a phosphatidylinositol bisphosphate breakdown resulting in an inositol trisphosphate production. These data suggested that substance P activated a phospholipase C and thus mediated its effects through the calcium-phospholipid pathway. To determine which neurokinin (NK) receptor was involved in the substance P response, we have used selective agonists of the different NK receptors and examined their effects on both inositol trisphosphate production and calcium movements. A selective NK-1 receptor agonist, [Sar9Met(O2)11]-substance P, evoked an [3H]inositol trisphosphate production and a rapid and transient 45Ca2+ efflux. On the other hand, selective NK-2 and NK-3 receptor agonists, [beta-Ala8]-NKA(4-10) and [MePhe7]-NKB, respectively, were without effect. We conclude that, in the rat parotid glands, only the NK-1 receptors are coupled to the calcium-phospholipid pathway. The C-terminal part of substance P appeared to be sufficient to stimulate this route because the C-terminal octapeptide, substance P(4-11), mimicked substance P effects on both inositol trisphosphate production and calcium movements. The NK-2 and NK-3 receptors, if present in the rat parotid glands, are not associated with the calcium-phospholipid pathway.     

PLCzeta(zeta): a sperm protein that triggers Ca2+ oscillations and egg activation in mammals

At fertilization in mammals, the sperm activates development by causing a prolonged series of intracellular Ca(2+) oscillations that are generated by increased production of inositol trisphosphate (InsP(3)). It appears that the sperm initiates InsP(3) generation via the introduction of a sperm factor into the egg after gamete membrane fusion. We recently identified a sperm-specific form of phospholipase C (PLC), referred to as PLCzeta(zeta). We review the evidence that PLCzeta represents the sperm factor that activates development of the egg and discuss the characteristics of PLCzeta that distinguish it from the somatic forms of PLC.