Inositol Bis Research Articles

Homomers of Kir.3.4 in atrial myocytes: their relevance to atrial fibrillation

The vagus slows the heart by raising potassium permeability and the activated K+ current – IK(ACh) – is carried by heteromers of the G-protein-gated inward rectifier potassium channel subunits Kir3.1 and Kir3.4 (for review, see Stanfield et al. 2002). However, in atrial myocytes only about half of Kir3.4 can be purified in heteromers with Kir3.1 and the remainder forms Kir3.4 homomers (Corey & Clapham, 1998). Quite what these homomeric channels do physiologically has been a puzzle: mice with Kir3.1 knocked out show mild tachycardia, which fails to respond to carbachol suggesting that Kir3.4 homomers do not contribute significantly to IK(ACh) (Bettahi et al. 2002). Clinical interest in the molecular biology of these channels is heightened by the need to manage atrial fibrillation, a common complication of heart failure and valvular disease. The dysrhythmia leads to fast, ‘irregularly irregular’ pacing of the ventricles, compromising cardiac output. In addition, blood is held in atria, and thrombus formation, particularly in the left auricle, increases risk of stroke. Atrial fibrillation of congestive heart failure (CHF) has often been managed with digoxin in the UK, and the drug has been held, on the basis of ECG monitoring, to have an effect via the vagus nerve, activating IK(ACh) (Krum et al. 1995). Atrial fibrillation (AF) is followed by electrophysiological remodelling of the heart, a process that is reversed after restoration of sinus rhythm (Brundel et al. 2001; Gaborit et al. 2005). In atrial myocytes, Kir3.1 expression is reduced relative to that of Kir3.4. Kir3.0 currents become constitutively active and the response to carbachol is reduced. The shortening of atrial action potentials may be corrected by the bee venom tertiapin, to which there is increased sensitivity (Dobrev et al. 2005), and use of derivatives of this venom has been seen as a possible method of management, mirroring use of the K+ channel blocker amiodarone. In this issue of The Journal of Physiology, Mintert et al. (2007) address some of these issues, using over-expression of Kir3.4 to mimic part of what happens in persistent AF. The homomeric channels produced show weaker inward rectification than is seen with Kir3.1/3.4 heteromers (the residues binding gating polyamines come from both Kir3.1 and 3.4, e.g. Stanfield et al. 2002). Unlike Kir3.1/3.4 heteromers, which must be gated open by Gβγ, these homomers are constitutively active. Nonetheless, FRET studies show homomers do interact with Gβγ. Kir3.0 channels require the membrane lipid phosphatidyl inositol bis phosphate (PIP2) as cofactor. Channels are thought to be activated by intracellular Na+ (Sui et al. 1998), Na+ binding to an aspartate residue in the C-terminus (Ho & Murrell-Lagnado, 1999) of Kir3.4. (The residue is Asn in Kir3.1.) By masking the negative charge of aspartate, Na+ aids the binding of PIP2. Surprisingly in view of this, the paper by Mintert et al. (2007) shows that IK(ACh) of atrial myocytes, carried by Kir3.1/3.4 heteromers, is not activated by internal Na+ at concentrations up to 60 mm; but Kir3.4 homomers are highly sensitive to Na+. The sensitivity to Na+ does not require activation through Gβγ since GTP-β-S has little inhibitory effect. Na+-activated currents are little inhibited by partial depletion of PIP2: a higher affinity probably means that PIP2 can bind from a depleted pool. Finally, channels are exquisitely sensitive to tertiapin-Q, affinity being at least 10-fold greater than that of Kir3.1/Kir3.4 heteromers. Do these findings concerning activation only of homomeric channels, which come to dominate over heteromers in atrial fibrillation, explain how digoxin controls AF? The drug might raise internal Na+ and activate Kir3.4 currents. In fact Mintert et al. (2007) are careful not to draw this conclusion, which would be controversial in the light of clinical literature describing the shortening of atrial action potentials as central to AF, with derivatives of tertiapin being seen as a solution to management. Further studies of this common dysrhythmia are sure to follow.

Rapid analysis of inositol phosphates.

Fast and simple analytical methods for the determination of inositol bis- to hexakisphosphates or only inositol hexakisphosphate in foods and feces are presented. The methods are both faster and simpler with regard to analytical detection and sample pretreatment as compared to previously reported methods. The samples are pretreated using extraction and centrifugal ultrafiltration and analyzed using high-performance ion chromatography (HPIC) with gradient or isocratic elution. The analytes are detected using ultraviolet detection after postcolumn reaction. The methods are efficient, highly selective, and appropriate for analyzing inositol phosphates in food and feces samples. The between- and within-day variances were generally below 8 and 5% (relative standard deviation), respectively, for the presented HPIC method with gradient elution.

Activation of the Na+/H+Exchanger is Required for Reperfusion-induced Ins(1,4,5)P3Generation

Post-ischemic reperfusion causes a change in inositol phosphate responses to norepinephrine from primary generation of inositol(1,4) bis phosphate (Ins(1,4)P2) to generation of inositol(1,4,5) tris phosphate (Ins(1,4,5)P3) that is required for the initiation of reperfusion arrhythmias. The current study was undertaken to investigate the role of Na+/H+exchange in facilitating this transient change in inositol phosphate response. Rat hearts were subjected to 20 min ischemia followed by 2 min reperfusion and Ins(1,4,5)P3content was measured by mass analysis or by anion-exchange HPLC following [3H]inositol labeling. Reperfusion caused generation of [3H]Ins(1,4,5)P3(1732±398 to 3103±214, cpm/g tissue, mean±S.E.M., n=5, P<0.01) and the development of arrhythmias. Inhibition of Na+/H+exchange, by reperfusing at pH 6.3 or by pretreating with HOE-694 (10 nM –3 μM) or HOE-642 (3 μM) prevented the [3H]Ins(1,4,5)P3generation, without causing any suppression of norepinephrine release. Increases in Ins(1,4,5)P3mass were similarly reduced by inhibition of Na+/H+exchange. Thus, activation of Na+/H+exchange is required for the enhanced Ins(1,4,5)P3response observed under reperfusion conditions, and prevention of Ins(1,4,5)P3generation may be an important contributor to the anti-arrhythmic actions of inhibitors of Na+/H+exchange.

Stimulation of respiratory burst by cyclocommunin in rat neutrophils is associated with the increase in cellular Ca 2+ and protein kinase C activity

In this study, the underlying mechanisms of stimulation by cyclocommunin, a natural pyranoflavonoid, of respiratory burst in rat neutrophils was investigated. Cyclocommunin evoked a concentration-dependent stimulation of superoxide anion (O 2 − ) generation with a slow onset and long lasting profile. The maximum response (16.4 ± 2.3 nmol O 2 − /10 min per 10 6 cells) was observed at 3–10 μM cyclocommunin. Cyclocommunin did not activate NADPH oxidase in a cell-free system. Cells pretreated with pertussis toxin or n-butanol did not affect the cyclocommunin-induced O 2 − generation. However, a protein kinase inhibitor staurosporine and EGTA greatly reduced the O 2 − generation caused by cyclocommunin. Treatment of neutrophils with phorbol 12-myristate 13-acetate (PMA), but not with formylmethionyl-leucyl-phenylalanine (fMLP), for 20 min significantly reduced the O 2 − generation following the subsequent stimulation of cells with cyclocommunin. Cyclocommunin did not affect the cellular mass of phosphatidic acid (PA). Neither the tyrosine kinase inhibitor, genistein, nor the p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, affected cyclocommunin-induced O 2 − generation. The enzyme activities of neutrophil cytosolic and membrane-associated protein kinase C (PKC) were both increased significantly with 100 μM cyclocommunin. The membrane-associated PKC-θ and PKC-β were increased following the stimulation of neutrophils with 30 and 100 μM cyclocommunin, respectively. Cyclocommunin reduced the [ 3H]phorbol 12,13-dibutyrate ([ 3H]PDB) binding to cytosolic PKC in a concentration-dependent manner. Cyclocommunin (≥3 μM) significantly evoked a slow and long lasting [Ca 2+] i elevation in neutrophils, and a phospholipase C (PLC) inhibitor U73122 greatly inhibited these Ca 2+ responses. Moreover, the increase in cellular inositol bis- and trisphosphate (IP 2 and IP 3) levels were observed in neutrophils stimulated with 30 μM cyclocommunin for 3 min. Collectively, these results indicate that the stimulation of respiratory burst by cyclocommunin is probably mediated by the synergism of PKC activation and [Ca 2+] i elevation in rat neutrophils.

The signal transduction mechanism involved in kazinol B-stimulated superoxide anion generation in rat neutrophils.

1. In this study, the underlying mechanism of stimulation of respiratory burst by kazinol B, a natural isoprenylated flavan, in rat neutrophils in vitro was investigated. 2. Kazinol B concentration-dependently stimulated the superoxide anion (O2*-) generation, with a lag but transient activation profile, in neutrophils but not in a cell-free system. The maximum response (13.2+/-1.4 nmol O2*- 10 min(-1) per 10(6) cells) was observed at 10 microM kazinol B. 3. Pretreatment of neutrophils with phorbol 12-myristate 13-acetate (PMA) or formylmethionyl-leucyl-phenylalanine (fMLP) significantly enhanced the O2*- generation following the subsequent stimulation of cells with kazinol B. 4. Cells pretreated with EGTA or a protein kinase inhibitor staurosporine effectively attenuated the kazinol B-induced O2*- generation. However, a p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 and a phosphoinositide 3-kinase (PI3K) inhibitor wortmannin had no effect on the kazinol B-induced response. 5. Kazinol B significantly stimulated [Ca2+]i elevation in neutrophils, with a lag and slow rate of rise activation profile, and this response was attenuated by a phospholipase C (PLC) inhibitor U73122. Kazinol B also stimulated the inositol bis- and trisphosphate (IP2 and IP3) formation with a 1 min lag time. 6. The membrane-associated PKC-alpha and PKC-theta but not PKC-iota were increased following the stimulation of neutrophils with kazinol B. It was more rapid and sensitive in the activation of PKC-theta than PKC-alpha by kazinol B. Kazinol B partially inhibited the [3H]phorbol 12,13-dibutyrate ([3H]PDB) binding to the neutrophil cytosolic PKC. 7. Neither the cellular mass of phosphatidic acid (PA) and phosphatidylethanol (PEt), in the presence of ethanol, nor the protein tyrosine phosphorylation were stimulated by kazinol B. In addition, the kazinol B-induced O2*- generation remained relatively unchanged in cells pretreated with ethanol or a tyrosine kinase inhibitor genistein. 8. Collectively, these results indicate that the stimulation of the respiratory burst by kazinol B is probably mediated by the synergism of PKC activation and [Ca2+]i elevation in rat neutrophils.

Thrombin receptor actions in neonatal rat ventricular myocytes.

Previous studies established that thrombin stimulates phosphoinositide hydrolysis and modulates contractile function in neonatal rat ventricular myocytes. The present study further defines the signaling pathways activated by the thrombin receptor and their role in thrombin's actions in cardiac myocytes. The thrombin receptor-derived agonist peptide (TRAP, a portion of the tethered ligand created by thrombin's proteolytic activity) stimulates the rapid and transient accumulation of inositol bis- and tris-phosphates (IP2 and IP3, respectively), which is followed by the more gradual and sustained accumulation of inositol monophosphate (IP1). TRAP elicits a larger and more sustained accumulation of IP1 than does thrombin. Thrombin and TRAP also activate mitogen-activated protein kinase (MAPK) in cultured neonatal rat ventricular myocytes. Differences in the kinetics and magnitude of thrombin- and TRAP-dependent inositol phosphate (IP) accumulation are paralleled by differences in the kinetics and magnitude of thrombin- and TRAP-dependent activation of MAPK. Pretreatment with phorbol 12-myristate 13-acetate (PMA) to downregulate protein kinase C (PKC) attenuates thrombin- and TRAP-dependent activation of MAPK, although small and equivalent effects of thrombin and TRAP to stimulate MAPK persist in PMA-pretreated cells. These results support the notion that the thrombin receptor activates MAPK through PKC-dependent pathways and that the incremental activation of MAPK by TRAP over that induced by thrombin is the consequence of enhanced activation through the PKC limb of the phosphoinositide lipid pathway. TRAP also increases the beating rate of spontaneously contracting ventricular myocytes and elevates cytosolic calcium in myocytes electrically driven at a constant basic cycle length. The effects of TRAP to modulate contractile function and elevate intracellular calcium are not inhibited by tricyclodecan-9-yl-xanthogenate (D609, to block TRAP-dependent IP accumulation) or pretreatment with PMA (to downregulate PKC). The TRAP-dependent rise in intracellular calcium also is not inhibited by verapamil or removal of extracellular calcium but is markedly attenuated by depletion of sarcoplasmic reticular calcium stores by caffeine. Patch-clamp experiments demonstrate that TRAP elevates intracellular calcium in cells held at a membrane potential of -70 mV. Taken together, these results support the conclusion that the thrombin receptor modulates contractile function by mobilizing intracellular calcium through an IP3-independent mechanism and that this response does not require activation of voltage-gated ion channels.

Neomycin and Spermine Have Similar Effects on Secretion and Phosphoinositide Metabolism in ATP-Depleted Permeabilized Adrenal Chromaffin Cells

To evaluate the requirement of bovine adrenal chromaffin cells for inositol phospholipids in secretion, the effects of neomycin and spermine on secretion and phosphoinositide metabolism were compared. Spermine and neomycin had virtually identical effects on secretion and phosphoinositide levels. Both polyamines 1) partially maintained secretion when added to permeabilized cells in the absence of ATP, 2) had no effect when added to cells in the presence of ATP and 3) inhibited secretion when present with the Ca 2+ stimulus. In the absence of ATP the enhancements to secretion due to incubation with either polyamine were associated with sustained levels of the polyphosphoinositides. The ability of spermine and neomycin to maintain secretion and the polyphosphoinositides in the absence of ATP supports the hypothesis that the maintenance of the polyphosphoinositides is necessary for secretion. Neomycin was found to inhibit Ca 2+ stimulated production of both inositol bis- and tris-phosphates while spermine was found to selectively inhibit inositol bis-phosphate production and had no effect on Ca 2+-stimulated inositol tris-phosphate production in permeabilized cells.

Multiple isomers of phosphatidyl inositol monophosphate and inositol bis- and trisphosphates from filamentous fungi

Multiple isomers of phosphatidyl inositol monophosphate and inositol bis- and trisphosphates from filamentous fungi

Interaction of vanadate with isolated rat parotid acini

1. In rat parotid acini, vanadate affects amylase secretion and intracellular messengers according to its concentration. 2. Low concentrations (in the micromolar range) concentrations of vanadate stimulate amylase secretion and potentiate the secretory effect of carbamylcholine, but inhibit the amylase release in response to isoproterenol. 3. Vanadate increases inositol phosphates (mono-, bis- and trisphosphate) and potentiate the stimulatory effect of carbachol on inositol bis- and trisphosphate. 4. Vanadate also decreases the intracellular cyclic AMP concentration and the increase in response to isoproterenol or forskolin. 5. High concentrations (in the millimolar range) of vanadate inhibit the increase in inositol phosphate induced by carbachol. 6. It is concluded that low concentrations of vanadate activate regulatory proteins coupled to phospholipase C and adenylate cyclase while high concentrations of vanadate inhibit inositol bisphosphatase.

Norepinephrine stimulates the direct breakdown of phosphatidyl inositol in rat tail artery.

When segments of rat tail artery were labeled with [3H]inositol and then stimulated with norepinephrine (NE), the inositol phosphates produced were primarily IP and IP2, together with a small but significant amount of Ins(1,4,5)P3 and a very small amount of Ins(1,3,4,5)P4. It has been unclear in many studies whether or not the relatively large levels of IP and IP2 produced in [3H]inositol-labeled tissue represent indirect products of phosphatidyl inositol(4,5)bis phosphate breakdown (through Ins(1,4,5)P3) or direct products of phosphatidyl inositol 4 monophosphate and phosphatidyl inositol breakdown. In order to answer this question tail artery segments were prelabeled with [3H]inositol and then permeabilized with beta escin and stimulated with norepinephrine and GTP gamma S, so that increases in IP, IP2, and Ins(1,4,5)P3 were still observed. If these permeable segments were stimulated with agonist in the presence of compounds known to inhibit Ins(1,4,5)P3 5-phosphatase, such as glucose 6P, (2,3)diphosphoglycerate, or Ins(1,4,5)P3, the levels of labeled Ins(1,4,5)P3 and labeled IP2 were increased, while the level of stimulated labeled IP was unchanged. This indicated that some of the IP2 and IP formed in these cells was produced from PIP2 but that some of these compounds might be formed from PIP or PI. When the isomers of inositol monophosphate, Ins 1P and Ins 4P, were separated by HPLC, it was shown that after prelabeled tail artery was stimulated by norepinephrine for periods of 1-2 min, the predominant isomer formed was Ins 4P, indicating either PIP2 or PIP as the source. However, after 5-20 min stimulation, both Ins 1P and Ins 4P were formed in equal amounts, suggesting that during sustained stimulation of smooth muscle PI itself was broken down directly. Therefore it appears that within 1-2 min of norepinephrine addition to vascular smooth muscle the bulk of the IP and IP2 produced are derived from PIP2 via IP3, while after 20 min of norepinephrine treatment much of the IP comes directly from PI. This suggests that the regulation of PLC in this tissue is more complicated than has been previously believed.

Calcium-sensitivity of inositol 1,4,5-trisphosphate metabolism in exocrine cells from the avian salt gland.

The generation of inositol phosphates upon muscarinic-receptor activation was studied in [3H]inositol-loaded exocrine cells from the nasal salt glands of the duck Anas platyrhynchos, and the metabolism of different inositol phosphates in vitro was studied in tissue homogenates, with particular reference to the possible interaction of changes in intracellular [Ca2+] ([Ca2+]i) with the metabolic processes. In intact cells, there was a rapid (within 15 s) generation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4, followed by an accumulation of their breakdown products, Ins(1,3,4)P3 and inositol bis- and monophosphates. Ca(2+)-sensitivity of the Ins(1,4,5)P3 3-kinase was demonstrated in tissue homogenates, with the rate of phosphorylation increasing 2-fold at free Ca2+ concentrations greater than 1 microM. However, addition of calmodulin or the presence of the calmodulin inhibitor W-7 (up to 100 microM) had no effect. 3-Kinase activity increased proportionally with the initial Ins(1,4,5)P3 concentration up to 1 microM, but a 10-fold higher substrate concentration produced only a doubling in the phosphorylation rate. Ins(1,3,4,5)P4 was dephosphorylated to Ins(1,3,4)P3, which accumulated in the homogenate assays as well as in intact cells. Depending on its concentration, Ins(1,3,4)P3 was phosphorylated [in part to Ins(1,3,4,6)P4] or dephosphorylated. To investigate the Ca(2+)-sensitivity of the 3-kinase in intact cells, excess quin2 was used to buffer the receptor-mediated transient changes in [Ca2+]i in [3H]inositol-loaded cells. These experiments revealed that increasing [Ca2+]i from less than 100 to approx. 400 nM (i.e. within the physiological range) has no effect on the partitioning of Ins(1,4,5)P3 metabolism (phosphorylation versus dephosphorylation) and on the accumulation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4. This indicates that activation of the 3-kinase by physiologically relevant Ca2+ concentrations may not play a major role in the generation of Ins(1,3,4,5)P4 signals upon receptor activation in these cells. The latter are mainly achieved by the receptor-mediated increase in Ins(1,4,5)P3 in the cell and its phosphorylation by the 3-kinase in a substrate-concentration-dependent manner.

Receptors and neurosecretory actions of endothelin in hypothalamic neurons.

Primary cultures of rat hypothalamic neurons were found to secrete the potent calcium-mobilizing and mitogenic peptide endothelin (ET) and to contain specific ET binding sites with higher affinity for ET-1 and ET-2 than ET-3. ET receptors of similar specificity were also identified in two gonadotropin-releasing hormone (GnRH) neuronal cell lines (GT1-1 and GT1-7). In both primary cultures and GnRH neurons, receptor binding of ETs led to marked and dose-dependent increases of inositol phosphates; inositol bis-, tris-, and tetrakisphosphates increased promptly, reached a peak within 2 min, and returned toward the steady-state levels during the next 10 min. ET-1 was more potent than ET-3 in mobilizing inositol phosphates, consistent with its greater affinity for the ET receptors in these cells. ET also stimulated GnRH secretion from perifused hypothalamic cultures and GnRH cell lines, with a sharp increase followed by a prompt decline to the basal level. These data show that ET is produced in the hypothalamus and acts through calcium-mobilizing ET receptors in normal and transformed secretory neurons to stimulate GnRH release. These actions of locally produced ETs upon GnRH-secreting neurons indicate that the vasoconstrictor peptides have the capacity to regulate neurosecretion and could participate in the hypothalamic control of anterior pituitary function and gonadotropin secretion.

Evidence for direct non-genomic effects of triiodothyronine on bone rudiments in rats: Stimulation of the inositol phosphate second messenger system

Thyroid hormones increase cytosolic free calcium by binding to plasma membrane receptors in several tissues. This calcium increase appears to initiate extranuclear effects in these tissues. Increases in cytosolic calcium are often a consequence of stimulation of inositol phosphate second messenger pathway. Several calcemic hormones act via this signal transduction route. Therefore we investigated the effects of the metabolically active T3 and the inactive analogues 3,5-diiodotyrosine and rT3 on the inositol phosphate pathway in fetal rat limb bone cultures prelabeled with [3H]myoinositol. Labelled inositol and inositol phosphates were separated by HPLC. There was a significant increase in the radioactivity in inositol bis- and trisphosphates after 1 min of exposure to 10(-7) mol/l T3. Stimulation was also observed at 10(-6) mol/l T3, but not at 10(-5) mol/l. Time course studies demonstrated a rapid effect of T3 on inositol phosphates within 30 seconds that lasted through 5 min. After 20 min incubation with T3, no increase was observed in inositol mono- and bisphosphates, and a decrease was seen in inositol trisphosphate. Pretreatment with indomethacin prevented these effects of T3. 3,5-diiodotyrosine and rT3 did not affect inositol phosphate metabolism. These results suggest the existence of plasma membrane-associated receptors for T3 in bone, in addition to the nuclear receptors demonstrated previously. The role of these receptors in the effects of thyroid hormones on bone remains to be established.

Histamine-induced inositol phosphate accumulation in HeLa cells: lithium sensitivity.

1. In the presence of 10 mM Li+ the histamine-stimulated accumulation of [3H]-inositol monophosphates [( 3H]-IP1) in HeLa cells prelabelled with [3H]-inositol increased over 10-20 min to a plateau level, which was normally maintained up to 60 min. Levels of [3H]-inositol bis- and trisphosphates [( 3H]-IP2 and [3H]-IP3) initially increased rapidly but declined to near basal levels by 20 min. 2. The same pattern of histamine-induced [3H]-IP1 accumulation was observed in cells in which [3H]-inositol was present 30 min before and during the incubation with histamine. Concentration-response curves for histamine measured in the presence of 10 mM Li+ were closely similar in cells prelabelled for 24 h with [3H]-inositol and in cells exposed to [3H]-inositol for only 30 min before addition of histamine, without removing the [3H]-inositol. The EC50 for histamine was 1.6 +/- 0.2 microM. 3. [3H]-IP1 accumulation induced by a sub-maximal concentration of histamine, 1 microM, also reached a plateau, but at a lower level than with 1 mM histamine. 4. Addition of 10 mM NaF at the plateau phase of [3H]-IP1 accumulation induced by 1 mM histamine resulted in a further increase in the level of [3H]-IP1. The level of [3H]-IP1 in the presence of histamine + NaF was 1.4 +/- 0.2 fold of that of the sum of the responses to histamine and NaF acting alone (basal levels subtracted). 5. Addition of 1 microM mepyramine at the plateau phase of [3H]-IP1 accumulation induced by 1 mM histamine in the presence of 10mM Li + resulted in a decline in the level of [3H]-IP1, implying that the metabolism of [3H]-IP1 is not completely blocked by 10mM Li' in HeLa cells. 6. Omission of the 15 min preincubation period with 10mM Li+ before stimulation with 100 microM histamine for 15 min resulted in an approximate halving of the level of [3H]-IP1, without any significant change in basal accumulation. Periods of preincubation with 10mM Li' longer than 15min did not produce any further increase in the level of [3H]-IP1 induced by histamine. 7. Basal and histamine-induced levels of [3H]-IP, increased as the concentration of Li+ was increased from 0 to 60mm, but the effect on the histamine-induced response was greater than on the basal level. Increasing the concentration of Li+ from 0 to 60 mm had only a small and mostly statistically insignificant effects on the levels of [3H]-1P2 and [3H]-1P3. The EC50 for histamine-induced [3H]-4P1 accumulation in the presence of 30mm Li + was 2.4 +/- 0.4 microM. 8. The results indicate that IP1 metabolism in HeLa cells is much less sensitive to Li+ than in mammalian brain. The plateau phase of histamine-induced [3H]-IP1 accumulation in the presence of 10mM Li+ thus represents a steady-state level. On a simple model the plateau level will be proportional to the rate of [3H]-IP1 formation at that time. The lower the concentration of Li' present, the better the approximation will be. The information obtained is thus not the same as when [3H]-1P, metabolism is completely blocked, in which case [3H]-IP1 accumulated can be used to calculate a mean rate of formation over the whole of the incubation period.

Progesterone-induced second messengers at the onset of meiotic maturation in the amphibian oocyte: Interrelationships between phospholipid N-methylation, calcium and diacylglycerol release, and inositol phospholipid turnover

The steady-state turnover in phospholipid N-methylation, 1,2-diacylglycerol and inositol phospholipids in prophase-arrested Rana pipiens oocytes was compared with changes occurring in these pathways immediately following progesterone induction of the first meiotic division. Oocytes were preincubated with [ 3H- methyl]methionine, [ 3H]glycerol, [ 3H] myo-inositol or [ 3H]arachidonic acid. Ca 2+ efflux was measured in oocytes preloaded with 45Ca 2+. Membrane phospholipids and cytosolic levels of radiolabeled 1,2-diacylglycerol (DAG), inositol bis- (InsP 2), tris- (InsP 3), and tetrakisphosphate (InsP 4) were monitored immediately following induction with progesterone. A transient increase in both N-methylation of ethanolamine phospholipids and in [ 3H]DAG coincides with a release of 45Ca 2+ from the oocyte surface during the first minute. At least 80% of the total phospholipid N-methylation is associated with the plasma membrane. 45Ca 2+ and [ 3H]DAG release occur prior to a rise in intracellular lnsP 3, the latter beginning 2–3 min after exposure to the hormone and reaching a maximum by 15–30 min. Progesterone induces rapid and successive changes in ethanolamine, choline, and inositol-containing phospholipids, which represent three of the four major phospholipid classes found in membranes. The maintenance of higher levels of DAG and InsP 3 during the first 90 min might be expected to sustain the previously observed increase in protein kinase C activity.

Inositol 1,4,5-trisphosphate activity in membranes isolated from amphibian skeletal muscle

The hydrolysis of [ 3H]inositol 1,4,5-trisphosphate by a soluble fraction and by isolated transverse tubule and sarcoplasmic reticulum membranes from frog skeletal muscle was studied. Transverse tubule membranes displayed rates of hydrolysis several-fold higher than those of sarcoplasmic reticulum and soluble fraction; K m and V max were 25.2 μM and 44.1 nmol/mg/min, respectively. Transverse tubule membranes sequentially hydrolyzed inositol trisphosphate to inositol bisphosphate, inositol 1-phosphate and inositol. Indicating that these membranes have inositol bis- and monophosphatases in addition to inositol trisphosphatase.

Activation of multiple signal transduction pathways by endothelin in cultured human vascular smooth muscle cells

Cellular responses to the vasoconstrictor peptide, endothelin, have been investigated in quiescent cultured human vascular smooth muscle cells (hVSMC). Endothelin caused intracellular alkalinization and activation of the protein synthetic enzyme S6-kinase, but such responses were not associated with any mitogenic effects of endothelin on hVSMC. In myo-[3H]inositol-prelabelled hVSMC endothelin elicited a rapid increase in inositol bis- and tris-phosphates and concomitant hydrolysis of polyphosphoinositol lipids. In [3H]arachidonate-prelabelled hVSMC endothelin promoted production of diacylglycerol, the early kinetics of which parallelled polyphosphoinositol lipid hydrolysis. Such phospholipase C activation by endothelin was sustained in hVSMC with accumulation of inositol polyphosphates being markedly protracted and the decay of diacylglycerol slow. Endothelin promoted extracellular release of [3H]arachidonate-labelled material from hVSMC which derived via deacylation of both phosphatidylinositol and phosphatidylcholine. This process was inhibited by phospholipase A2 and lipoxygenase inhibitors, but insensitive to phospholipase C and cyclooxygenase inhibitors. Endothelin-induced activation of phospholipase C and phospholipase A2 signal transduction pathways (EC50 approximately 5-8 nM for both) in hVSMC apparently proceed in an independent parallel manner rather than a sequential one.

ENDOTHELIN STIMULATES INOSITOL PHOSPHATE ACCUMULATION IN RAT RIGHT AND LEFT ATRIA: A COMPARISON WITH NORADRENALINE

1. The effect of endothelin on phosphatidylinositol turnover in rat atria was investigated by measuring the generation of inositol phosphates following [3H]-inositol labelling. 2. In the presence of 10 mmol/L LiCl, endothelin caused dose-dependent increases in the accumulation of inositol mono, bis and trisphosphates which were maximal at 10(-6) mol/L endothelin. The dose-response relationship was similar in right and left atria, but right atria showed a higher maximal inositol phosphate response. 3. Endothelin produced a rapid and transient stimulation of inositol trisphosphate accumulation, which peaked at 15 s followed by a slower increase which continued linearly past 20 min. 4. The time course of inositol trisphosphate release under noradrenaline stimulation showed a similar profile but the maximum stimulation was smaller than endothelin. 5. As with endothelin, responses to noradrenaline also were higher in right atria compared with left atria. 6. These data demonstrate that endothelin receptors in rat atria are coupled to the stimulation of the phosphatidylinositol turnover pathway in an essentially similar manner to alpha 1-adrenoceptors. The phosphatidylinositol pathway may be important in mediating the reported cardiac actions of endothelin.

Aluminum fluoride reveals a phosphoinositide system within the suprachiasmatic region of rat hypothalamus

The phosphoinositide (PI) transduction system has proven to be of major importance in several regions of mammalian brain. In this report, we examined in rats whether a PI system is present in the hypothalamic suprachiasmatic nuclei (SCN), the site of a biological clock that generate circadian rhythms. Autoradiographic localization of phorbol ester binding revealed moderate levels of protein kinase C, a component of the PI system, in the SCN. Hypothalamic explants containing SCN showed substantial incorporation of [ 3H]myoinositol into lipids. A AlF 4 −, a non-specific activator of G proteins, produced a dose-dependent increase in inositol monophosphate (IP 1) levels in the explants in calcium-free medium, with a maximum increase of 216% of control at 50 mM NaF. Medium containing 1.8 mM calcium stimulated a similar increase in IP 1 levels, but the stimulatory effects of AlF 4 − and calcium were not additive, so that the effect of AlF 4 − was obscured in medium containing calcium. AlF 4 − stimulated accumulation of IP 1, as well as inositol bis-, and triphosphate, over a 40-min time course in the presence and absence of lithium (10 mM LiCl). Lithium, a known inhibitor of phosphatases in the inositol phosphate recycling pathway, raised levels of all 3 inositol phosphates in SCN explants both at baseline (without AlF 4 −) and after 30 minAlF 4 − stimulation. The results show the existence of a lithium-sensitive PI system within the suprachiasmatic region of the rat hypothalamus.

The effect of non-NMDA antagonists and phorbol esters on excitatory amino acid stimulated inositol phosphate formation in rat cerebral cortex

The effects of various modulators of excitatory amino acid stimulated inositol phosphate (IP) formation were investigated in slices of rat cerebral cortex. Both quisqualic acid (Quis) and ibotenic acid (Ibo) stimulated IP formation in a dose-dependent manner. Quis (0.3mM) stimulated the rapid formation of inositol bis, tris and tetrakis-phosphates, with a slower linear rise in the monophosphate which plateaued after 20 min. The responses to both Ibo (0.3 mM) and Quis (0.3 mM) were dose-dependently inhibited by phorbol dibutyrate (PDBu); the Ibo response was particularly sensitive to PDBu, with a 90% inhibition of the response at 1 ?M and an IC(50) of 200 nM; Quis stimulated IP formation was less sensitive with a 50% inhibition observed at 10 ?M PDBu. PMA (1 ?M) and dioctanoylglycerol (30 ?M) also inhibited Ibo-stimulated IP formation though 4-?-phorbol (10 ?M) was ineffective. The inhibition by PDBu was reversed with staurosporine (10 ?M) and polymyxin (10 ?M), and both protein kinase C inhibitors potentiated the Ibo response in the absence of PDBu. The putative Quis-kainate receptor antagonists 6,7-dinitroquinoxaline-2,3-dione and 6-cyano-7-nitroquinoxaline-2,3-dione did not inhibit the responses to either Quis or Ibo at a concentration of 100 ?M. Both responses were, however, inhibited by dl-2-amino-4-phosphonobutyric acid and O- phospho- l -serine (IC(50)s were 1-2 mM), and also by the putative Quis receptor agonist dl-?-amino-3-hydroxy-5-methyl-4-isoxalone propionic acid (AMPA; IC(50) 10?M). Our data suggest that excitatory amino acid stimulated IP formation in rat cerebral cortex is via a site distinct from previously defined amino acid receptors.