Stimulation of cytosolic free calcium and inositol phosphates by prostaglandins in cultured rat mesangial cells

We studied the effects of four products of arachidonate cyclo-oxygenation on a phospholipase C-dependent signal transduction system in cultured rat glomerular mesangial cells. PGF2 alpha, PGE2 and the thromboxane A2/endoperoxide analogue U-46619 rapidly increased cytosolic free Ca2+, measured in monolayers loaded with the fluorescent intracellular probe fura-2. Peak responses were dose-dependent and unaffected by chelation of extracellular Ca2+, indicating release from internal stores. The thromboxane A2-receptor antagonist SQ 27,427 selectively inhibited responses to U-46619. The PGI2 analogue Iloprost had no effect on cytosolic Ca2+. PGF2 alpha, PGE2 and U-46619 also stimulated accumulation of total inositol phosphates during 15 min incubations. We conclude that phospholipase C activation mediates the effects of certain eicosanoids on the glomerular mesangium.     

The labelling of polyphosphoinositides with [32P]Pi and the accumulation of inositol phosphates in vasopressin-stimulated hepatocytes.

When hepatocytes were incubated with [32P]Pi, the kinetics for the labelling of the monoester phosphate groups of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate were similar to each other and slightly slower than that for the labelling of the gamma-phosphate of ATP. Analysis of the water-soluble 3H-labelled materials derived from [3H]inositol-labelled hepatocytes revealed that, in addition to inositol and its mono-, bis- and tris-phosphates (Ins, InsP, InsP2 and InsP3), these cells contained two unidentified radioactive compounds which co-eluted with InsP on anion-exchange chromatography. When [3H]inositol-labelled hepatocytes were stimulated with 0.23 microM-vasopressin in the presence of 10 mM-Li+, there was an accumulation of radioactivity in InsP, InsP2 and InsP3 but not in Ins or the two unidentified compounds. Further analysis of these inositol phosphates by h.p.l.c. revealed that vasopressin also stimulates the accumulation of inositol tetrakisphosphate (InsP4) in these cells. Vasopressin-stimulated InsP and InsP2 accumulations were maximal in the presence of 1-10 mM-Li+ but InsP3 accumulation continued to increase up to 50 mM-Li+. Accumulated inositol phosphates were retained within the cell. Li+ from 1 to 50 mM did not influence the extent of vasopressin-stimulated inositol lipid degradation in hepatocytes. In the absence of Li+, radioactivity in vasopressin-stimulated hepatocytes accumulated almost entirely in free inositol. The vasopressin-stimulated accumulation of inositol phosphates in the presence of 10 mM-Li+ was abolished by a V1-vasopressin antagonist. Inositol phosphate accumulation was not influenced by ionophore A23187, dimethyl sulphoxide or indomethacin.     

Hydrolysis of [Leu]enkephalin by chick brain in vitro.

Using high-performance liquid chromatography with electrochemical detection to measure substrate disappearance and metabolite accumulation following addition of [Leu]enkephalin to samples prepared from chick brain in vitro, the following were found: 1. [Leu]enkephalin hydrolysis by whole forebrain homogenates is almost solely attributable to aminopeptidase MII activity. 2. [Leu]enkephalin hydrolysis by whole forebrain P2 membrane fractions is attributable to both aminopeptidase MII and dipeptidyl carboxypeptidase activity. 3. Differences are apparent in both [Leu]enkephalin disappearance and Tyr-Gly-Gly accumulation in P2 membrane fractions, but not in homogenate fractions, prepared from several regions of the chick brain.     

Interactions between inositol phosphates and cytosolic free calcium following bradykinin stimulation in cultured human skin fibroblasts

The inositol triphosphate (IP3) that results from hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) is generally accepted to be responsible for the mobilization of intracellular calcium. However, some studies suggest that low concentrations of agonists elevate cytosolic free calcium concentration ([Ca2+]i) without IP3 formation. Thus, in the present studies, a comparison of the temporal response of inositol phosphates (IP3, IP2 and IP) and [Ca2+]i to a wide range of bradykinin concentrations was used to examine the relation of these two signal transduction events in cultured human skin fibroblasts (GM3652). In addition, the effects of alterations in internal or external calcium on the response of these second messengers to bradykinin were determined. Bradykinin stimulated accumulation of inositol phosphates and a rise of [Ca2+]i in a time- and dose-dependent manner. Decreasing the bradykinin concentration from 1 microM to 0.1 microM increased the time until the IP3 peak, and when the bradykinin concentration was reduced to 0.01 microM IP3 was not detected. [Ca2+]i was examined under parallel conditions. As the bradykinin concentration was reduced from 1 microM to 0.01 microM, the time to reach the peak of [Ca2+]i increased progressively, but the magnitude of the peak was unaltered. These two second messengers were variably dependent on external calcium. Although the bradykinin-stimulated initial spike of [Ca2+]i did not depend on extracellular calcium, the subsequent sustained levels of [Ca2+]i were abolished in calcium free medium. The bradykinin-stimulated inositol phosphate formation was not dependent on the extracellular calcium nor on the elevation of [Ca2+]i that was produced with Br-A23187. These results demonstrate that bradykinin-induced IP3 formation can be independent of [Ca2+]i and of external calcium, whereas changes in [Ca2+]i are partially dependent on external calcium.     

Relationship between inositol polyphosphate production and the increase of cytosolic free Ca2+ induced by vasopressin in isolated hepatocytes

Addition of vasopressin to rat hepatocytes prelabeled with myo-[2-3H]inositol resulted in a very rapid decrease [3H]phosphatidylinositol 4,5-bisphosphate (Ptd-Ins-4,5-P2) which was paralleled by increases of up to 3-fold in the levels of [3H]inositol trisphosphate (Ins-P3) and [3H]inositol bisphosphate (Ins-P2). Increases of [3H]inositol phosphate (Ins-P) were not detected until about 5 min after hormone addition. These data indicate that the major pathway for hormone-induced lipid breakdown in liver is through a phosphodiesterase for PtdIns-4,5-P2 and that decreases of phosphatidylinositol are a secondary result of increased PtdIns-4,5-P2 resynthesis. Using the fluorescent Ca2+ indicator Quin 2, cytosolic free Ca2+ increased from 160 nM to about 400 nM after vasopressin addition to hepatocytes and preceded the conversion of phosphorylase b to a. Half-maximal and maximal increases of cytosolic free Ca2+ and phosphorylase a activity were observed at 0.2 and 1 nM vasopressin, respectively. The dose-response curve for the initial rate of cytosolic free Ca2+ increase was very similar to those obtained for the initial rates of Ins-P3 production and PtdIns-4,5-P2 breakdown. Pretreatment of hepatocytes with Li+ caused a 3--4-fold potentiation of vasopressin-induced elevations of Ins-P, Ins-P2, and Ins-P3, with half-maximal effects at 0.5, 1, and 5 mM, respectively. The calculated maximal concentrations of Ins-P3 in cells treated with 20 nM vasopressin were 10 and 30 microM, respectively, without and with Li+. Lithium did not affect the initial rate of inositol polyphosphate production or Ca2+ mobilization. The increase of Ins-P3 which correlated with peak cytosolic free Ca2+ elevation was about 0.6 microM. In a saponin-permeabilized hepatocyte preparation, Ins-P3 (1 microM) caused Ca2+ release from a vesicular, ATP-dependent Ca2+ pool. The data presented here suggest that Ins-P3 may be a second messenger for the mobilization of intracellular Ca2+ by hormones in liver.     

Changes in cytosolic free calcium concentration in isolated rat parotid cells by cholinergic and beta-adrenergic agonists

The alteration in the concentration of cytosolic free calcium ([Ca2+]i) in isolated rat parotid cells caused by autonomic agents was directly measured using the Ca-sensitive fluorescent probe, quin2. [Ca2+]i of unstimulated cells was estimated to be 162.7 +/- 3.2 nM in normal medium. Carbachol (CCh) and isoproterenol (ISP) caused a rapid rise in [Ca2+]i in a dose-dependent manner. Maximum increases in [Ca2+]i induced by CCh and ISP were approximately 100% and 25% of resting level, respectively. In Ca-free medium, CCh produced a small, rapid rise in [Ca2+]i, followed by a slow decay and a return to resting level within 3-4 min, while all doses of ISP tested failed to change [Ca2+]i. These results suggest that CCh mobilizes Ca2+ from both extracellular and intracellular pools and then results in a rise in [Ca2+]i, whereas ISP may slightly mobilize only the extracellular Ca pool.     

Incorporation of [3H]inositol into phosphoinositides and inositol phosphates by rabbit blastocysts

Preimplantation rabbit embryos collected at the early morula stage were cultured to blastocysts in the presence of [³H]inositol. The blastocysts were lysed, and both the aqueous and lipid portions were analysed for incorporated radioactivity. Thin-layer chromatographic separation of the lipid portion indicated that [³H]inositol was incorporated into phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate. HPLC anion-exchange chromatography indicated that [³H]inositol was incorporated into inositol phosphates, including the two second messengers, inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate, and also inositol monophosphate and inositol 1,4-bisphosphate. These results provide evidence that rabbit blastocysts may have an active phosphatidylinositol second messenger system, which may be responsive to intrauterine factors or intraembryonic paracrine factors. © 1993 Wiley-Liss, Inc.     

Conformational preferences of [Leu5]enkephalin in biomimetic media. Investigation by 1H NMR

The conformation of [Leu5]enkephalin has been studied by 1H-NMR spectroscopy in media more like the actual environment in which the agonist-receptor interaction takes place than water, i.e. in three cryoprotective mixtures (dimethylformamide/water, methanol/water and ethylene glycol/water), in aqueous SDS and in two neat solvents, dimethylformamide and acetonitrile, whose dielectric constants (36.7 and 37.5) are intermediate between that of water and that of the lipid phase. In all cases examined, contrary to the studies in water or dimethylsulfoxide, we were able to detect numerous nuclear Overhauser effects, indicating that the media employed favour well-defined structures and/or reduce the internal motions of the peptide. Data from both organic solvents and cryoprotective mixtures suggest a 4----1 beta turn as the most probable structure of [Leu5]enkephalin in solution, whereas in SDS/H2O micelles the structural picture appears completely different, suggesting the presence of a 5----2 beta turn. The existence of two different preferred conformations of enkephalins may possibly be related to their ability to be effective towards both mu and delta opioid receptors.     

Synthesis and biological activity of [Leu5]enkephalin derivatives containing D-glucose

The synthesis of some [Leu5]enkephalin derivatives is described in which D-glucose has been linked to the opioid pentapeptide through the ester bond involving the carboxyl function at the C-terminal with C-1 or C-6 of the D-glucopyranose moiety. Enkephalin derivatives were assayed for opioid activity and found to be full agonists in bioassays based on inhibition of electrically evoked contractions of the guinea pig ileum (GPI) and of the mouse vas deferens (MVD). The obtained results suggest that the opioid activity of the tested glucoconjugates depend upon the ester bond position in the molecule. Whereas 1-O conjugate 5 was somewhat more potent than [Leu5]enkephalin in the GPI assay, the 6-O conjugates, with the exception of 1-O-benzyl derivative 11, were considerably less potent. All enkephalin derivatives were delta-receptor selective; in particular, the acetylated analog 8 was three times more delta-receptor selective than [Leu5]enkephalin.     

Persistent inhibition by inositol 1,4,5-trisphosphate of oxalate-dependent 45calcium accumulation in permeable guinea-pig hepatocytes

Guinea-pig hepatocytes whose plasma membranes were rendered permeable by treatment with saponin, accumulated 45calcium in the presence of potassium oxalate and ATP. The uptake was linear with time for up to one hour when high-capacity EGTA buffers were used (5mM). In the presence of a supra-maximal concentration of inositol 1,4,5-trisphosphate, under conditions minimising metabolism of this calcium-mobilising messenger, 45calcium accumulation was inhibited by about 40% for a period of one hour. Electron microscopic examination of the cells, revealed the presence of electron dense precipitates. Electron microprobe analysis of the precipitates indicated that they constituted the majority of the oxalate-dependent calcium uptake. The precipitates were located throughout the non-nuclear regions of the cells. Cells treated with inositol 1,4,5-trisphosphate contained fewer precipitates, but high cell-to-cell variability prevented conclusions as to the precise location of the pool sensitive to inositol 1,4,5-trisphosphate. These results support the previous contention that a fraction of endoplasmic reticulum is completely emptied of calcium by maximal concentrations of inositol 1,4,5-trisphosphate, while another fraction is insensitive to this action. In addition, these findings indicate that the pool of intracellular calcium on which inositol 1,4,5-trisphosphate acts is oxalate-permeable, and that the calcium-releasing action of inositol 1,4,5-trisphosphate does not desensitise within one hour.     

Comparison of inositol phosphates accumulation induced by different effectors in rat thyroid slices.

Rat thyroid slices were submitted to different effectors and hormones in order to investigate their action on the phosphatidylinositol metabolism. Fluoride and vanadate induced a clear increase of the inositol phosphates with half maximal stimulation at 7 mM and 8 mM respectively. The inositol bisphosphate (IP2) and inositol trisphosphate (IP3) accumulation induced by vanadate was relatively higher than that observed in the case of fluoride stimulation. Carbachol stimulated also the generation of inositol phosphates with half maximal activation at 2.5 x 10(-6) M. In the same conditions, no significant effect on inositol phosphates production could be detected by the action of TSH or TRH.     

ATP stimulates inositol phosphates accumulation and calcium mobilization in a primary culture of rat aortic myocytes

The effects of extracellular ATP on phosphoinositide metabolism and intracellular Ca2+ concentration were studied in a primary culture of rat aortic myocytes. ATP increases the level of inositol phosphates, the putative second messenger for Ca2+ mobilization. No saturation of inositol phosphates accumulation is obtained (up to 10(-2) M ATP). Under the same conditions, ATP rapidly mobilizes intracellular Ca2+ in fura-2 loaded myocytes. The mobilization of intracellular Ca2+ is dose-dependent (maximal at 10(-4) M ATP), and is not affected by addition of EGTA. It is concluded that the receptors mediating the cytosolic increase of Ca2+ are of the P2-purinoceptor subtype. The physiological functions of these receptors are not presently known.     

Control of cytosolic free calcium by intracellular organelles in bovine adrenal glomerulosa cells. Effects of sodium and inositol 1,4,5-trisphosphate

The regulation of cytosolic free Ca2+ concentration ([Ca2+]c) by intracellular organelles was studied in permeabilized bovine adrenal glomerulosa cells. Two compartments, with distinct characteristics, were able to pump Ca2+. A first pool, sensitive to ruthenium red and presumably mitochondrial, required respiratory chain substrates to maintain [Ca2+]c around 700 nM. Ca2+ efflux from this compartment was activated by Na+ (ED50 = 5 mM). Inositol 1,4,5-trisphosphate (IP3) had no effect on this pool. A second nonmitochondrial pool required ATP to lower [Ca2+]c to about 200 nM and released Ca2+ transiently upon addition of IP3. When the two systems were allowed to work simultaneously, the nonmitochondrial pool regulated [Ca2+]c and IP3 released Ca2+ in a concentration-dependent manner (EC50 = 0.6 microM). Under these conditions the mitochondria seemed Ca2+ depleted. Upon repeated stimulations with IP3, a marked attenuation of the response was observed. This phenomenon was due to Ca2+ sequestration by a nonmitochondrial IP3-insensitive pool. Neither dantrolene (200 microM) nor 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (10 microM) were able to abolish IP3-induced Ca2+ release, though both compounds efficiently inhibited aldosterone production in intact cells stimulated with angiotensin II (10 nM) or K+ (12 mM). These results suggest that in permeabilized adrenal glomerulosa cells: the nonmitochondrial pool is responsible for buffering [Ca2+]c and for releasing Ca2+ in response to IP3; at resting [Ca2+]c levels, the mitochondria appear Ca2+ depleted; when [Ca2+]c rises above their set point, the mitochondria accumulate Ca2+ as a function of [Na+]c; 4) the mitochondria are not involved in the desensitization mechanism of the response to IP3.     

Actions of inositol phosphates on Ca2+ pools in guinea-pig hepatocytes.

In permeabilized hepatocytes, inositol 1,4,5-trisphosphate, inositol 2,4,5-trisphosphate and inositol 4,5-bisphosphate induced rapid release of Ca2+ from an ATP-dependent, non-mitochondrial vesicular pool, probably endoplasmic reticulum. The order of potency was inositol 1,4,5-trisphosphate greater than inositol 2,4,5-trisphosphate greater than inositol 4,5-bisphosphate. The Ca2+-releasing action of inositol 1,4,5-trisphosphate is not inhibited by high [Ca2+], nor is it dependent on [ATP] in the range of 50 microM-1.5 mM. These results suggest a role for inositol 1,4,5-trisphosphate as a second messenger in hormone-induced Ca2+ mobilisation, and that a specific receptor is involved in the Ca2+-release mechanism.     

Pertussis toxin blocks activin A-induced production of inositol phosphates in rat hepatocytes.

The present study was conducted to examine an involvement of G protein in the action of activin A in rat parenchymal liver cells. Activin A induced a dose-dependent increase in inositol phosphates in cells prelabelled with [3H]inositol. The effect of activin A was completely blocked by pretreatment of the cells with pertussis toxin. In contrast, pertussis toxin had little effect on angiotensin II-induced production of inositol phosphates. Both activin A and angiotensin II inhibited glucagon-mediated production of cAMP. Pretreatment of the cells with pertussis toxin blocked the inhibition induced by both activin A and angiotensin II. In permeabilized cells, activin A augmented production of inositol phosphates. Activin-mediated production of inositol trisphosphate was enhanced by GTP-gamma S and was attenuated by GDP-beta S. These results suggest that a pertussis toxin-sensitive G protein(s) may be involved in the action of activin A in hepatocytes.     

Kinetics of chymotrypsin- and papain-catalysed synthesis of [leucine]enkephalin and [methionine]enkephalin.

The proteinase-catalysed synthesis of [Leu]enkephalin and [Met]enkephalin was studied kinetically. N alpha-t-Butoxycarbonyl-amino acids and peptides or their ethyl esters served as acyl donors, and amino acid phenylhydrazides were used as acyl acceptors. Initial-velocity measurements of alpha-chymotrypsin-catalysed peptide synthesis gave rise to kinetic patterns that are compatible with a ping-pong mechanism modified by a hydrolytic branch. Initial-rate and alternative-substrate inhibition patterns for papain-controlled peptide-bond formation are consistent with a sequential ordered mechanism with the acyl donor as the obligatory first substrate. On the basis of the observed kinetic features, reaction mechanisms are proposed for chymotrypsin- and papain-catalysed peptide synthesis that inversely equal those describing the pathways of proteolysis. The respective initial-velocity expressions for bireactant systems are given, along with the numerical values of the corresponding kinetic parameters.     

Synthesis and biological activity of a lysine-containing cyclic analog of [Leu5]enkephalin

The cyclic analogue of [Leu5]enkephalin--cyclo (Lys-Tyr-Gly-Gly-Phe-Leu) and two corresponding linear hexapeptides with lysine residue attached to the N- or C-terminus of the molecule have been synthesized by classical methods of peptide chemistry. The addition of lysine residue to the N-terminus of cyclization of the molecule reduce the interaction of these analogues with both central and peripheral opiate receptors. The addition of lysine residue to the C-terminus of the molecule through the epsilon-amino group does not affect the interaction of the analogue with mu-receptors but reduces approximately tenfold its affinity for delta-receptors. All three analogues have analgesic potency similar to that of [Leu5]enkephalin as assayed after intracisternal administration to mice.     

Regulation of cytosolic free calcium concentration by intrasynaptic mitochondria.

By the use of digitonin permeabilized presynaptic nerve terminals (synaptosomes), we have found that intrasynaptic mitochondria, when studied "in situ," i.e., surrounded by their cytosolic environment, are able to buffer calcium in a range of calcium concentrations close to those usually present in the cytosol of resting synaptosomes. Adenine nucleotides and polyamines, which are usually lost during isolation of mitochondria, greatly improve the calcium-sequestering activity of mitochondria in permeabilized synaptosomes. The hypothesis that the mitochondria contributes to calcium homeostasis at low resting cytosolic free calcium concentration ([Ca2+]i) in synaptosomes has been tested; it has been found that in fact this is the case. Intrasynaptic mitochondria actively accumulates calcium at [Ca2+]i around 10(-7) M, and this activity is necessary for the regulation of [Ca2+]i. When compared with other membrane-limited calcium pools, it was found that depending on external concentration the calcium pool mobilized from mitochondria is similar or even greater than the IP3- or caffeine-sensitive calcium pools. In summary, the results presented argue in favor of a more prominent role of mitochondria in regulating [Ca2+]i in presynaptic nerve terminals, a role that should be reconsidered for other cellular types in light of the present evidence.     

Injection of inositol trisphosphorothioate into Limulus ventral photoreceptors causes oscillations of free cytosolic calcium

Limulus ventral photoreceptors contain calcium stores sensitive to release by D-myo-inositol 1,4,5 trisphosphate (InsP3) and a calcium-activated conductance that depolarizes the cell. Mechanisms that terminate the response to InsP3 were investigated using nonmetabolizable DL-myo-inositol 1,4,5 trisphosphorothioate (InsPS3). An injection of 1 mM InsPS3 into a photoreceptor's light-sensitive lobe caused an initial elevation of cytosolic free calcium ion concentration (Cai) and a depolarization lasting only 1-2 s. A period of densensitization followed, during which injections of InsPS3 were ineffective. As sensitivity recovered, oscillations of membrane potential began, continuing for many minutes with a frequency of 0.07-0.3 Hz. The activity of InsPS3 probably results from the D-stereoisomer, since L-InsP3 was much less effective than InsP3. Injections of 1 mM InsP3 caused an initial depolarization and a period of densensitization similar to that caused by 1 mM InsPS3, but no sustained oscillations of membrane potential. The initial response to InsPS3 or InsP3 may therefore be terminated by densensitization, rather than by metabolism. Metabolism of InsP3 may prevent oscillations of membrane potential after sensitivity has recovered. The InsPS3-induced oscillations of membrane potential accompanied oscillations of Cai and were abolished by injection of ethyleneglycol-bis (beta-aminoethyl ether)-N,N'-tetraacetic acid. Removal of extracellular calcium reduced the frequency of oscillation but not its amplitude. Under voltage clamp, oscillations of inward current were observed. These results indicate that periodic bursts of calcium release underly the oscillations of membrane potential. After each burst, the sensitivity of the cell to injected InsP3 was greatly reduced, recovering during the interburst interval. The oscillations may, therefore, result in part from a periodic variation in sensitivity to a constant concentration of InsPS3. Prior injection of calcium inhibited depolarization by InsPS3, suggesting that feedback inhibition of InsPS3-induced calcium release by elevated Cai may mediate desensitization between bursts and after injections of InsPS3.