alpha(1)-adrenergic stimulation mediates Ca(2+)-dependent inositol phosphate formation through the alpha(1B)-like adrenoceptor subtype in adult rat cardiac myocytes.

We studied the effects of increased Ca(2+) influx on alpha(1)-adrenoceptor-stimulated InsP formation in adult rat cardiac myocytes. We further examined if such effects could be mediated through a specific alpha(1)-adrenoceptor subtype. [(3)H]InsP responses to adrenaline were dependent on extracellular Ca(2+) concentration, from 0.1 microM to 2 mM, and were completely blocked by Ca(2+) removal. However, in cardiac myocytes preloaded with BAPTA, a highly selective calcium chelating agent, Ca(2+) concentrations higher than 1 microM had no effect on adrenaline-stimulated [(3)H]InsP formation. Taken together these results suggest that [(3)H]InsP formation induced by alpha(1)-adrenergic stimulation is in part mediated by increased Ca(2+) influx. Consistent with this, ionomycin, a calcium ionophore, stimulated [(3)H]InsP formation. This response was additive with the response to adrenaline stimulation implying that different signaling mechanisms may be involved. In cardiac myocytes treated with the alpha(1B)-adrenoceptor alkylating agent, CEC, [(3)H]InsP formation remained unaffected by increased Ca(2+) concentrations, a pattern similar to that observed when intracellular Ca(2+) was chelated with BAPTA. In contrast, addition of the alpha(1A)-subtype antagonist, 5'-methyl urapidil, did not affect the Ca(2+) dependence of [(3)H]InsP formation. Neither nifedipine, a voltage-dependent Ca(2+) channel blocker nor the inorganic Ca(2+) channel blockers, Ni(2+) and Co(2+), had any effect on adrenaline stimulated [(3)H]InsP, at concentrations that inhibit Ca(2+) channels. The results suggest that in adult rat cardiac myocytes, in addition to G protein-mediated response, alpha(1)-adrenergic-stimulated [(3)H]InsP formation is activated by increased Ca(2+) influx mediated by the alpha(1B)-subtype.     

Formation and metabolism of inositol 1,3,4,5-tetrakisphosphate in liver

The inositol lipid pools of isolated rat hepatocytes were labeled with [3H]myo-inositol, stimulated maximally with vasopressin and the relative contents of [3H]inositol phosphates were measured by high performance liquid chromatography. Inositol 1,4,5-trisphosphate accumulated rapidly (peak 20 s), while inositol 1,3,4-trisphosphate and a novel inositol phosphate (ascribed to inositol 1,3,4,5-tetrakisphosphate) accumulated at a slower rate over 2 min. Incubation of hepatocytes with 10 mM Li+ prior to vasopressin addition selectively augmented the levels of inositol monophosphate, inositol 1,4-bisphosphate, and inositol 1,3,4-trisphosphate. A kinase was partially purified from liver and brain cortex which catalyzed an ATP-dependent phosphorylation of [3H]inositol 1,4,5-trisphosphate to inositol 1,3,4,5-tetrakisphosphate. Incubation of purified [3H]inositol 1,3,4,5-tetrakisphosphate with diluted liver homogenate produced initially inositol 1,3,4-trisphosphate and subsequently inositol 1,3-bisphosphate, the formation of which could be inhibited by Li+. The data demonstrate that the most probable pathway for the formation of inositol 1,3,4,5-tetrakisphosphate is by 3-phosphorylation of inositol 1,4,5-trisphosphate by a soluble mammalian kinase. Degradation of both compounds occurs first by a Li+-insensitive 5-phosphatase and subsequently by a Li+-sensitive 4-phosphatase. The prolonged accumulation of both inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate in vasopressin-stimulated hepatocytes suggest that they have separate second messenger roles, perhaps both relating to Ca2+-signalling events.     

Metabolism of inositol phosphates in alpha 1-adrenoceptor-stimulated and homogenized cardiac myocytes of adult rats.

Previous studies demonstrated the accumulation of inositol mono- and poly-phosphates in carbamoylcholine-stimulated cultured cardiac ventricular myocytes of adult rats [Berg, Guse & Gercken (1989) Biochim. Biophys. Acta 1010, 100-107]. Stimulation with noradrenaline (50 microM) in the presence of propranolol (10 microM) led to a time-dependent pattern of inositol mono- and poly-phosphates in cultured cardiac-ventricular myocytes. Ins(1,4,5)P3 and Ins(1,3,4,5)P4 increased in a rapid initial phase. The degradation products of Ins(1,4,5)P3, namely Ins(1,4)P2 and Ins(4)P, accumulated between 1 and 15 min. Ins(1,3,4,5)P4 was rapidly dephosphorylated to Ins(1,3,4)P3, which was then metabolized to Ins(1,3)P2 and Ins(3,4)P2. The last two InsP2 isomers and their degradation products, Ins(1)P and Ins(3)P (determined as an enantiomeric mixture), increased at extended stimulation periods. To confirm the pathway of Ins(1,3,4,5)P4 degradation, homogenates of isolated ventricular myocytes were incubated with [3H]INs(1,3,4,5)P4. The subsequent products were Ins(1,3,4)P3, Ins(3,4)P2, Ins(1,3)P2 and InsP. The effect of noradrenaline was antagonized by prazosin (0.1 microM), but not by yohimbine (0.1 microM), indicating phosphoinositidase activation via the alpha 1-adrenoceptor.     

Rapid formation of inositol 1,3,4,5-tetrakisphosphate following muscarinic receptor stimulation of rat cerebral cortical slices.

Carbachol stimulation of muscarinic receptors in rat cortical slices prelabelled with myo-[2-3H]inositol caused the rapid formation of a novel inositol polyphosphate. Evidence derived from its chromatographic behaviour, and from the structure of the products formed in partial dephosphorylation experiments, suggests that it is probably D-myo-inositol 1,3,4,5-tetrakisphosphate. An enzyme in human red cell membranes specifically removes the 5-phosphate from it to form inositol 1,3,4-trisphosphate. It is suggested that inositol 1,3,4,5-tetrakisphosphate is likely to be a second messenger, and that it is the precursor of inositol 1,3,4-trisphosphate and possibly of inositol 1,4,5-trisphosphate.     

Modulation of ICa-L by alpha1-adrenergic stimulation in rat ventricular myocytes

We found when L-type calcium current (ICa-L) was recorded with the perforated patch-clamp method in rat ventricular myocytes that bath application of phenylephrine (with propranolol) evoked a biphasic response characterized by an initial transient suppression followed by a sustained potentiation. The transient suppression occurred 30-60 s after phenylephrine perfusion and reached peak inhibition at approximately 2 min. The biphasic modulation of ICa-L was also elicited by methoxamine, and the effects of phenylephrine were blocked by prazosin, indicating that the responses were mediated through alpha1-adrenoceptors. Pretreatment of cells with H7 (100 micromol/L), a broad-spectrum protein kinase inhibitor that inhibits both protein kinase C and A, eliminated potentiation but did not affect transient suppression. The transient suppression occurred concurrently with the acceleration of the fast component of ICa-L inactivation. Depletion of intracellular Ca2+ stores by ryanodine plus caffeine or thapsigargin eliminated the transient suppression. When ICa-L was recorded with whole-cell patch-clamp and with 0.05 mmol/L EGTA in the pipette solution to allow intracellular Ca2+ to fluctuate, phenylephrine evoked a transient suppression as in the perforated patch recordings. Heparin, a specific blocker of IP3 (inositol 1,4,5-trisphosphate) receptors, eliminated the phenylephrine-induced transient suppression of ICa-L when added to the pipette solution. Intensive chelation of intracellular Ca2+ by 5 mmol/L BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) in the pipette solution also eliminated the phenylephrine-induced transient suppression of ICa-L. We conclude that transient increase in the concentration of intracellular calcium ([Ca2+]i) caused by Ca2+ release from intracellular stores underlies the transient suppression of ICa-L, whereas the potentiation of ICa-L is a result of activation of protein kinases.     

Interconversion of inositol (1,4,5)-trisphosphate to inositol (1,3,4,5)-tetrakisphosphate and (1,3,4)-trisphosphate in permeabilized adrenal glomerulosa cells is calcium-sensitive and ATP-dependent

The metabolism of [3H]inositol (1,4,5)-trisphosphate was followed in permeabilized bovine adrenal glomerulosa cells. At low Ca++ concentration (pCa = 7.2), more than 90% of [3H]inositol (1,4,5)-trisphosphate had disappeared within 2 min, while two other metabolites, [3H]inositol (1,3,4)-trisphosphate and [3H]inositol (1,3,4,5)-tetrakisphosphate appeared progressively. At higher Ca++ concentrations (pCa = 5.7 and 4.8), the formation of these two metabolites was markedly increased, but completely abolished if the medium was ATP-depleted. The peak levels for the generation of [3H]inositol (1,3,4,5)-tetrakisphosphate (1 min) preceded those of [3H]inositol (1,3,4)-trisphosphate and were closely correlated. These results suggest that, in adrenal glomerulosa cells, the isomer inositol (1,3,4)-trisphosphate is generated from inositol (1,4,5)-trisphosphate via a calcium-sensitive and ATP-dependent phosphorylation/dephosphorylation pathway involving the formation of inositol (1,3,4,5)-tetrakisphosphate.     

Design and synthesis of biotinylated inositol 1,3,4,5-tetrakisphosphate targeting Grp1 pleckstrin homology domain

A bifunctional molecule containing biotin and d-myo-inositol 1,3,4,5-tetrakisphosphate was synthesized. This molecule was designed on the basis of X-ray structure of the complex of d-myo-inositol 1,3,4,5-tetrakisphosphates, Ins(1,3,4,5)P(4), and Grp1 PH (general receptor of phosphoinositides pleckstrin homology) domain for the application to the widely employed biotin-avidin techniques. The building block of inositol moiety was synthesized starting with myo-inositol and assembled with the biotin-linker moiety through a phosphate linkage. The equilibrium dissociation constant K(D) of biotinylated Ins(1,3,4,5)P(4) binding of original Grp1 PH domain was 0.14 μM in pull-down analysis, which was comparable to that of unmodified Ins(1,3,4,5)P(4). Furthermore, biotinylated Ins(1,3,4,5)P(4) had an ability to distinguish Grp1 PH domain from PLCδ(1) PH domain. Thus, biotinylated Ins(1,3,4,5)P(4) retained the binding affinity and selectivity of original Grp1 PH domain, and realized the intracellular Ins(1,3,4,5)P(4) despite a tethering at the 1-phosphate group of inositol.     

alpha(1D)-Adrenoceptors do not contribute to phosphoinositide hydrolysis in adult rat cardiac myocytes

The thermodynamic parameters of the alkaline transition of beef heart ferricytochrome c have been measured through direct electrochemistry experiments carried out at variable pH and temperature in the presence of different sulfate concentrations. Sulfate is known to bind specifically to cytochrome c in a sequential manner at two surface sites. The effects of such a specific binding reflect on the thermodynamics of the transition and can be satisfactorily interpreted within the frame of the Debye-Hückel theory with simple electrostatic considerations. In particular, the increase in the thermodynamic pKa values (extrapolated to I = 0) upon sulfate binding turns out to be a fully enthalpic effect which can be accounted for by considering the coulombic effects of the formation of ionic couple(s) on the protein surface. This study also shows that the apparent pKa values at finite ionic strength are only moderately affected by the nature of the anion in solution, and differences tend to vanish at high ionic strength.     

Regulation of MAPK pathways in response to purinergic stimulation of adult rat cardiac myocytes

We investigated the activation of mitogen-activated protein kinases (MAPKs) pathways by purinergic stimulation in cardiac myocytes from adult rat hearts. ATPS increased the phosphorylation (activation) of the extracellular signal regulated kinase 1 and 2 (ERK1/2) and p38 MAPK. ERK1/2 and p38 MAPK activation was differential, ERK1/2 being rapid and transient while that of p38 MAPK slow and sustained. Using selective inhibitors, activation of ERK1/2 was shown to involve protein kinase C and MEK1/2 while that of p38 MAPK was regulated by both protein kinase C and protein kinase A. Furthermore, we show that purinergic stimulation induces the phosphorylation of the MAPK downstream target, mitogen- and stress-activated protein kinase 1 (MSK1), in cardiac myocytes. The time course of MSK1 phosphorylation closely follows that of ERK activation. Inhibitors of the ERK and p38 MAPK pathways were tested on the phosphorylation of MSK1 at two different time points. The results suggest that ERKs initiate the response but both ERKs and p38 MAPK are required for the maintenance of the complete phosphorylation of MSK1. The temporal relationship of MSK1 phosphorylation and cPLA₂ translocation induced by purinergic stimulation, taken together with previous findings, is an indication that cPLA₂ may be a downstream target of MSK1.     

The role of alpha 1-adrenergic stimulation in inositol phosphate metabolism during post-ischaemic reperfusion.

The aim of this study was to elucidate the mechanism of enhanced inositol phosphate metabolism during reperfusion. Inositol phosphate stores were prelabelled by perfusing isolated rat hearts for 1 h with [3H]inositol (1.5 microCi/ml). LiCl (10 mM) and prazosin (0.3 microM) were subsequently added 15 min before (i) 20 min control perfusion; (ii) 20 min normothermic ischaemic cardiac arrest (NICA); (iii) 20 min NICA followed by 1 min reperfusion. The ventricles were freeze-clamped before determination of isotopical incorporation of [3H]inositol into the inositol phosphates (Dowex anion exchange chromatography) and InsP3 levels (Amersham InsP3 assay system). In addition, noradrenaline release into the perfusate was also assessed (HPLC and electrochemical detection). The results showed: (i) increased noradrenaline release into the perfusate immediately after the onset of reperfusion; (ii) significant depression of [3H]inositol incorporation into inositol phosphates and InsP3 levels after 20 min NICA; (iii) reperfusion caused an immediate significant increase in isotopical incorporation of [3H]inositol into inositol phosphates as well as InsP3 levels; (iv) the alpha 1-adrenergic blocker, prazosin (0.3 microM), completely inhibited the reperfusion-induced increase in inositol phosphate metabolism. These observations suggested that increased alpha 1-adrenergic receptor stimulation by noradrenaline might be responsible for the stimulation of ventricular inositol phosphate metabolism during postischaemic reperfusion.     

Kinetics of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate generation in dog-thyroid primary cultured cells stimulated by carbachol

The action of carbachol on the generation of inositol trisphosphate and tetrakisphosphate isomers was investigated in dog-thyroid primary cultured cells radiolabelled with [3H]inositol. The separation of the inositol phosphate isomers was performed by reverse-phase high pressure liquid chromatography. The structure of inositol phosphates co-eluting with inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] standards was determined by enzymatic degradation using a purified Ins(1,4,5)P3/Ins(1,3,4,5)P4 5-phosphatase. The data indicate that Ins(1,3,4,5)P4 was the only [3H]inositol phosphate which co-eluted with a [32P]Ins(1,3,4,5)P4 standard, whereas 80% of the [3H]InsP3 co-eluting with an Ins(1,4,5)P3 standard was actually this isomer. In the presence of Li+, carbachol led to rapid increases in [3H]Ins(1,4,5)P4. The level of Ins(1,4,5)P3 reached a peak at 200% of the control after 5-10 s of stimulation and fell to a plateau that remained slightly elevated for 2 min. The level of Ins(1,3,4,5)P4 reached its maximum at 20s. The level of inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] increased continuously for 2 min after the addition of carbachol. Inositol-phosphate generation was also investigated under different pharmacological conditions. Li+ largely increased the level of Ins(1,3,4)P3 but had no effect on Ins(1,4,5)P3 and Ins(1,3,4,5)P4. Forskolin, which stimulates dog-thyroid adenylate cyclase and cyclic-AMP accumulation, had no effect on the generation of inositol phosphates. The absence of extracellular Ca2+ largely decreased the level of Ins(1,3,4,5)P4 as expected considering the Ca2(+)-calmodulin sensitivity of the Ins(1,4,5)P3 3-kinase. Staurosporine, an inhibitor of protein kinase C, increased the levels of Ins(1,4,5)P3, Ins(1,3,4,5)P4 and Ins(1,3,4)P3. This supports a negative feedback control of diacyglycerol on Ins(1,4,5)P3 generation.     

Mass measurements of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate in a neuronal cell line stimulated with bradykinin: inositolphosphate response shows desensitization.

In a neuronal cell line (108CC15, NG108-15) the levels of inositol 1,4,5-trisphosphate (InsP3) and inositol 1,3,4,5-tetrakisphosphate (InsP4), as measured by receptor binding assays, rise transiently after stimulation with bradykinin (EC50 approx. 150 nM). Maximal InsP3 level of 354 pmol/mg protein (15-fold basal level) is obtained at 10-15 s after addition of bradykinin, the InsP4 level rises maximally to 78 pmol/mg protein (14-fold basal level) at 20-30 s. In a rat glioma cell line, bradykinin (2 microM) causes a fast 6-fold increase in InsP3 and InsP4 levels. In the neuronal cells the bradykinin-dependent rise of the inositolphosphate levels is diminished with reduced extracellular Ca2+ concentration. However, depletion of internal Ca2+ stores does not affect the bradykinin-induced rise in InsP3 and InsP4 levels. Homologous desensitization to bradykinin occurs in the signal transduction pathway already at the production of inositolphosphates, since after a 2 min stimulation with bradykinin the rise in cellular masses of InsP3 and InsP4, inducible by a following second bradykinin stimulus, is substantially reduced.     

Structure of the PH domain from Bruton's tyrosine kinase in complex with inositol 1,3,4,5-tetrakisphosphate

BACKGROUND: The activity of Bruton's tyrosine kinase (Btk) is important for the maturation of B cells. A variety of point mutations in this enzyme result in a severe human immunodeficiency known as X-linked agammaglobulinemia (XLA). Btk contains a pleckstrin-homology (PH) domain that specifically binds phosphatidylinositol 3,4,5-trisphosphate and, hence, responds to signalling via phosphatidylinositol 3-kinase. Point mutations in the PH domain might abolish membrane binding, preventing signalling via Btk. RESULTS: We have determined the crystal structures of the wild-type PH domain and a gain-of-function mutant E41K in complex with D-myo-inositol 1,3,4,5-tetra-kisphosphate (Ins (1,3,4,5)P4). The inositol Ins (1,3,4,5)P4 binds to a site that is similar to the inositol 1,4,5-trisphosphate binding site in the PH domain of phospholipase C-delta. A second Ins (1,3,4,5)P4 molecule is associated with the domain of the E41K mutant, suggesting a mechanism for its constitutive interaction with membrane. The affinities of Ins (1,3,4,5)P4 to the wild type (Kd = 40 nM), and several XLA-causing mutants have been measured using isothermal titration calorimetry. CONCLUSIONS: Our data provide an explanation for the specificity and high affinity of the interaction with phosphatidylinositol 3,4,5-trisphosphate and lead to a classification of the XLA mutations that reside in the Btk PH domain. Mis-sense mutations that do not simply destabilize the PH fold either directly affect the interaction with the phosphates of the lipid head group or change electrostatic properties of the lipid-binding site. One point mutation (Q127H) cannot be explained by these facts, suggesting that the PH domain of Btk carries an additional function such as interaction with a Galpha protein.     

Potassium channels regulated by inositol 1,3,4,5-tetrakisphosphate and internal calcium in DDT1 MF-2 smooth muscle cells

This study was carried out to determine the intracellular components responsible for the transmembrane current evoked by stimulation of H1-histaminergic receptors in DDT1 MF-2 smooth muscle cells. Histamine elicited an outward current that was reversed below the K+ equilibrium potential and passed voltage-independent K+ channels. A histamine concentration-dependent rise in outward current and in cytoplasmic-free Ca2+ with similar time courses was observed. The histamine-induced current was not found after depletion of internal Ca2+ stores, suggesting a coupling between internal Ca2+ and K+ current. The time course of the initial increase in inositol (1,4,5)-trisphosphate (Ins (1,4,5)P3) caused by histamine differs from that of the internal Ca2+ response. However, a significant concentration-dependent increase in inositol (1,3,4,5)-tetrakisphosphate (Ins (1,3,4,5)P4) was seen during the whole stimulating period. The role of internal Ca2+, Ins (1,4,5)P3, and Ins (1,3,4,5)P4 on the outward current was also examined by the addition of these substances directly to the cytoplasm. Internal application of Ca2+ increased the amplitude and duration of the histamine-induced current whereas internal EGTA suppressed the outward current. Internal Ins (1,4,5)P3 did not affect the histamine-induced K+ current, Ins (1,3,4,5)P4 inhibited the outward current, and the combination of Ins (1,3,4,5)P4 and Ca2+ abolished this response. The noradrenaline response evoked under normal conditions is not reflected by a change in transmembrane current or a change in Ins (1,3,4,5)P4 but is associated with an increase in Ins (1,4,5)P3 and internal Ca2+. Stimulation of alpha 1-adrenoceptors, however, also evoked an outward current after the addition of Ins (1,3,4,5)P4 intracellularly. It is concluded that K+ channels, carrying the histamine outward current, are activated from the combined action of internal Ca2+ and Ins (1,3,4,5)P4.     

Transient inositol (1,4,5) trisphosphate accumulation under vasopressin stimulation in WRK1 cells: correlation with intracellular calcium mobilization

In the rat mammary tumoral cell line (WRK1 cells), vasopressin was previously described to stimulate a phospholipase C. In this study, we have analysed the effect of vasopressin both on intracellular calcium mobilization and on the accumulation of inositol phosphates. Maximal concentration of vasopressin simultaneously induces an accumulation of Ins(1,4,5)P3 and a rise of intracellular calcium concentration. Both these two phenomena are transient and exhibit similar kinetics. A sustained accumulation of InsP2, Ins(1,3,4)P3 and InsP are observed later. Yet no stimulation of InsP4 can be objectified. These results indicate that Ins(1,4,5)P3 is the major inositol phosphate involved in intracellular calcium mobilization.     

Characterization of a high-affinity Ins-P4 (inositol 1,3,4,5-tetrakisphosphate) receptor from brain by an anti-peptide antiserum

From a high-affinity Ins-P₄ (inositol 1,3,4,5-P₄) receptor purified from pig cerebellum, digested with the protease Lys C peptide sequences were obtained. Synthetic peptide-3 (19 amino acid residues) was used to generate an antiserum. Reaction of the affinity-purified antibodies with the purified pig receptor protein in ELISA or Western blot was completely inhibited by peptide-3. In cerebellar membranes, the antibodies clearly recognized the 42 kDa Ins-P₄ receptor protein and two additional proteins (25 kDa, 37 kDa) which still have to be identified. The anti-peptide antibodies could selectively immunoprecipitate the Ins-P₄ receptor protein. The antiserum was used (i) to demonstrate that in brain from different species (human, pig, beef, rat, mouse and sheep) a similar 42 kDa Ins-P₄ receptor protein is contained, and (ii) to obtain indications for the existence of a related soluble form of the 42 kDa Ins-P₄ receptor besides the membrane-associated receptor.     

G beta gamma counteracts G alpha(q) signaling upon alpha(1)-adrenergic receptor stimulation

In rat neonatal myocytes, a constitutively active G alpha(q) causes cellular injury and apoptosis. However, stimulation of the alpha(1)-adrenergic receptor, one of the G(q) protein-coupled receptors, with phenylephrine for 48 h causes little cellular injury and apoptosis. Expression of the G beta gamma-sequestering peptide beta ARK-ct increases the phenylephrine-induced cardiac injury, indicating that G beta gamma released from G(q) counteracts the G alpha(q)-mediated cellular injury. Stimulation with phenylephrine activates extracellular signal-regulated kinase (ERK) and Akt, and activation is significantly blunted by beta ARK-ct. Inhibition of Akt by inhibitors of phosphatidylinositol 3-kinase increases the cellular injury induced by phenylephrine stimulation. In contrast to the inhibition of Akt, inhibition of ERK does not affect the phenylephrine-induced cardiac injury. These results suggest that G beta gamma released from G(q) upon alpha(1)-adrenergic receptor stimulation activates ERK and Akt. However, activation of Akt but not ERK plays an important role in the protection against the G alpha(q)-induced cellular injury and apoptosis.