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PubMed2000

Overexpression of Na(+)-dependent myo-inositol transporter gene in mouse lens led to congenital cataract.

PURPOSE: Maintaining appropriate osmotic pressure is essential for maintaining lens transparency. This study was performed to investigate whether high levels of myo-inositol, one of the major organic osmolytes in the lens, would lead to cataract development. METHODS: Transgenic mouse lines carrying the bovine Na(+)-dependent myo-inositol transporter (bSMIT) cDNA under the control of the mouse alphaA-crystallin promoter were generated. RESULTS. Increased bSMIT expression was accompanied by increased myo-inositol level in the lens and increased uptake of (3H) myo-inositol by the lens in culture. The transgenic mice developed observable cataract under normal rearing conditions beginning at 2 to 8 weeks of age, and the severity of cataract development was correlated to the level of bSMIT gene expression and lens myo-inositol accumulation. For transgenic mouse line 3352, heterozygous mice did not develop cataract, whereas homozygous ones did. Prenatal feeding of heterozygous 3352 mice with high myo-inositol diet led to cataract development, indicating that cataract development was not merely due to a nonspecific effect of SMIT overexpression. Introducing aldose reductase overexpressing transgene into heterozygous 3352 mice also led to cataract development, indicating that this type of cataract is primarily due to osmotic stress. CONCLUSIONS: The present results indicate that high levels of myo-inositol and sorbitol in the lens contribute to cataract development. This is a useful model to study the role of osmotic stress in cataractogenesis during lens development.

Aldehyde Reductase↗
PubMed1992

Neurotransmitters and neuropeptides stimulate inositol phosphates and intracellular calcium in cultured human nonpigmented ciliary epithelium.

The effects of several neurotransmitters and neuropeptides on the inositol phosphate/diacylglycerol pathway were examined in human nonpigmented ciliary epithelial cells. Maximal stimulation of inositol phosphate formation by vasopressin (approximately 3-fold), carbachol (approximately 2-fold) and histamine (approximately 5-fold) was observed only after cells had been confluent for at least six days. In contrast, a response to bombesin (approximately 3-fold) declined with extended time in confluent culture. Inositol monophosphate, inositol bisphosphate, and inositol trisphosphate all were stimulated by these agonists. Dose-response studies showed a close correlation between the EC50s of the different agonists when elevation of inositol phosphates was compared to stimulation of intracellular Ca2+, with the exception of bombesin. Preliminary pharmacologic characterization of the receptors for vasopressin, carbachol, and bombesin provided rank order of potencies for selective agonists and antagonists. The data suggest that the muscarinic receptor on human NPE cells is the M3 subtype, whereas the vasopressin receptor, as defined by its linkage to the inositol phosphate/diacylglycerol pathway, is the V1 subtype.

Analgesics↗
PubMed1990

The effect of dopamine D1 and D2 receptor agonists on inositol phosphate turnover in rat striatal slices.

There is a lot of controversy in the literature about the role of dopamine D1 and D2 receptor stimulation on the turnover of phosphoinositols, and phosphoinositols are one of the important second messenger. In order to resolve this controversy, the effect of dopamine receptor stimulation on turnover of phosphoinositols was studied by estimation of the accumulation of individual labelled inositols in rat striatal slices which were prelabelled with [3H]myoinositol. Incubation of the prelabelled striatal slices with 1 microM of quinpirole, D2 specific agonist or with 1 microM of SKF-38393, D1 specific agonist, did not affect the accumulation of basal level of either inositol monophosphate, or inositol biphosphate, or inositol triphosphate. In addition, in conclusion of D1 specific antagonist cis-flupentixol or D2 specific antagonist sulpiride did not affect the basal levels of inositol phosphates. The activity of enzyme phospholipase-C which produces these inositol phosphates was also measured in rat striatal membrane. Incubation of rat striatal membrane with either agonist quinpirole or SKF-38393 did not change the basal level of phospholipase C. Our data thus indicate that occupation of dopamine receptors did not affect the inositol phosphate system in rat striatum.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗
PubMed1990

Chinese hamster ovary cell mutants defective in myo-inositol transport.

By means of an in situ colony autoradiographic assay for the incorporation of [14C]inositol into the trichloroacetic acid-insoluble fraction, we have isolated a mutant of cultured Chinese hamster ovary cells defective in inositol transport, named mutant 648. Through comparison of the inositol uptake activity of 648 cells with that of the parental cells with various concentrations of inositol and sodium, it has been demonstrated that Chinese hamster ovary cells possess a sodium-dependent transport system for inositol, and that 648 cells lack this system. The sodium-dependent uptake is inhibited by 2,4-dinitrophenol and ouabain, and the intracellular concentration of inositol exceeds the extracellular concentration during the uptake period, indicating that it is active transport, at least partially driven by the sodium gradient generated by Na+,K(+)-ATPase. The apparent Km for inositol has been estimated to be 12.0 microM. It is inhibited by hyperglycemic concentration of D-glucose in a competitive fashion.

Animals↗
PubMed1989

Chemoattractant receptor promotion of Ca2+ influx across the plasma membrane of HL-60 cells. A role for cytosolic free calcium elevations and inositol 1,3,4,5-tetrakisphosphate production.

UNLABELLED: The mechanisms by which the chemotactic peptide formyl-methyl-leucyl-phenyl-alanine stimulates Ca2+ influx across the plasma membrane were investigated in the human promyelocytic cell line HL-60, induced to differentiate with dimethyl sulfoxide. Ca2+ influx was determined: (a) from the initial rate of Mn2+ influx, apparent from the quenching of intracellular quin2 or fura-2 fluorescence; (b) from the rate of the elevation of cytosolic free calcium, [Ca2+]i, upon readdition of Ca2+ to cells previously stimulated in the absence of extracellular Ca2+. [3H]Inositol tris-, tetrakis-, and pentakisphosphates were analyzed by a high performance liquid chromatography procedure which was optimized for the separation of inositol tetrakisphosphates, yielding three predominant isomers: inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), inositol 1,4,5,6-tetrakisphosphate, and inositol 1,3,4, 6-tetrakisphosphate. Both the kinetics and agonist dose dependence of Ca2+ influx stimulation correlated closely with the corresponding receptor-mediated variations of [Ca2+]i either in the presence or in the absence of extracellular Ca2+. Of the different inositol phosphates determined in parallel and under the same conditions, accumulation of [3H]Ins(1,3,4,5)P4 correlated best with Ca2+ influx both temporally and in its dose dependence in the presence or in the absence of extracellular Ca2+; inositol 1,3,4-trisphosphate was also correlated but to a lesser extent. Attenuations of [Ca2+]i elevations by decreasing extracellular Ca2+ or by increasing the cytosolic Ca2+ buffering capacity with quin2 led to parallel inhibition of Ca2+ influx and Ins(1,3,4,5)P4 production. IN CONCLUSION: 1) activation of Ca2+ influx by formyl-methionyl-leucyl-phenylalanine depends on the elevation of [Ca2+]i, the latter being initiated by Ca2+ mobilization from intracellular stores; 2) Ins(1,3, 4,5)P4 is a strong candidate for maintaining receptor-mediated activation of Ca2+ influx in differentiated HL-60 cells.

Aminoquinolines↗
PubMed1989

[Abnormal inositol phospholipid metabolism as a main factor causing pericyte drop-out in diabetic retinopathy].

The biochemical basis of pericyte loss in early diabetic retinopathy is still an open question. In studies on the mechanism by which retinal pericytes degenerate, we first established a bovine retinal capillary pericyte (BRCP) cell line. Subcultured BRCP grown in high (10-40 mmol/L) glucose media were used as an experimental model. We found that high concentrations of glucose suppress the mitotic rate and cell birth rate of cultured BRCP. High concentrations of glucose inhibit myo-inositol transport and result in decreased intracellular myo-inositol content. This inhibition can be partially reversed by sorbinil, an aldose reductase inhibitor (ARI). Myo-inositol is a precursor of inositol phospholipids (IPLs), whose metabolism is responsible for a number of signal transduction processes. Phosphoinositidase (PIase) cleaves the phosphodiester bond of phosphotidylinositol 4,5 diphosphate (PIP2) to produce two second messengers, inositol trisphosphate (IP3) and diacylglycerol (DG). Further experiments showed that IP3 and DG synergistically activate BRCP proliferation in vitro. High concentrations of glucose altered the formation of both IPLs and inositol phosphate esters (IPEs) in an organ culture of retinal microvessels. This alteration can be reversed by adding either high concentrations of myo-inositol or ARI to the medium. PIase activity was attenuated to 82% or 55% when glucose in the growth medium was increased from 5 to 15 or 30 mmol/L, respectively. When IPLs from BRCP were analyzed by HPLC and TLC, we observed the reduction of three IPLs, including the substrate of PIase, PIP2.(ABSTRACT TRUNCATED AT 250 WORDS)

Aldehyde Reductase↗
PubMed1989

Epidermal growth factor alters metabolism of inositol lipids and activity of protein kinase C in mouse embryo palate mesenchyme cells.

Epidermal growth factor (EGF) stimulated mouse embryo palate mesenchyme (MEPM) cells (1) to incorporate [32P]O4(3-) into phosphatidylinositol (PI), phosphatidylcholine, and phosphatidic acid over a period of 60 min; 2) to incorporate [32P]O4(3-) into polyphosphoinositides as a function of time; and 3) to incorporate [32P]O4(-3) into PI, only, as a function of concentration when the period of stimulation was kept short. EGF stimulated the release of radiolabeled inositol phosphates from MEPM cells that had been radiolabeled with [3H]myoinositol. The release of inositol 1-phosphate was sustained over a period of at least 60 min, whereas the release of inositol 1,4-bisphosphate and inositol trisphosphate peaked during the first 10 min of stimulation. EGF also stimulated phosphorylation of an Mr 80,000 protein whose pI, phosphopeptide map, and phosphoamino acid pattern were identical to those of an Mr 80,000 protein phosphorylated in response to phorbol 12-myristate 13-acetate. Mobilization or metabolism of arachidonic acid was not stimulated under the same conditions that permitted EGF to alter inositol lipid metabolism. We interpret these data to mean that 1) in contrast to the findings with some cell lines, alterations in inositol lipid metabolism may be part of the signalling mechanism for EGF in embryonic cells; 2) EGF is capable of activating inositol-dependent signalling pathways leading to activation of protein kinase C in MEPM cells; and 3) mobilization and metabolism of arachidonic acid are not an inherent part of this signalling mechanism.

Animals↗
PubMed1989

Inhibition of protein kinase C by staurosporine promotes elevated accumulations of inositol trisphosphates and tetrakisphosphate in human platelets exposed to thrombin.

We have examined regulation by protein kinase C (Ca2+/phospholipid-dependent enzyme) of thrombin-induced inositol polyphosphate accumulation in human platelets. When platelets are exposed to thrombin for 10 s, the protein kinase C inhibitor staurosporine causes inositol phosphate elevations over control values of 2.7-fold (inositol 1,4,5-trisphosphate (Ins(1,4,5)P3], 1.9-fold (inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4], and 1.2-fold (inositol 1,3,4-trisphosphate). In the same period, phosphatidic acid and diacylglycerol are unaffected. The myosin light chain kinase inhibitor ML-7 has no effect on inositol phosphate accumulations. Staurosporine does not inhibit Ins(1,4,5)P3 3-kinase and 5-phosphomonoesterase activities in saponin-permeabilized platelets incubated with exogenous Ins(1,4,5)P3 unless the platelets have been exposed to thrombin and protein kinase C is consequently activated. The protein kinase C agonist beta-phorbol 12,13-dibutyrate increases the Vmax of the 3-kinase 1.8-fold, with little effect on Km. Our results provide strong evidence for a role for protein kinase C in regulating inositol phosphate levels in thrombin-activated platelets. We propose that endogenously activated protein kinase C removes Ins(1,4,5)P3 by stimulating both 5-phosphomonoesterase and Ins(1,4,5)P3 3-kinase. Initial activation of phospholipase C does not appear to be affected by such protein kinase C. Inhibition of protein kinase C by staurosporine decreases 5-phosphomonoesterase activity. The resulting elevated Ins(1,4,5)P3, as substrate for Ins(1,4,5)P3 3-kinase, promotes production of Ins(1,3,4,5)P4, which also may accumulate through decreased 5-phosphomonoesterase activity and elevated Ca2+ levels. These factors apparently counteract the inhibitory effect on 3-kinase, yielding a net increase in Ins(1,3,4,5)P4.

Alkaloids↗
PubMed1989

Insulin-like growth factor I stimulates inositol phosphate accumulation, a rise in cytoplasmic free calcium, and proliferation in cultured porcine thyroid cells.

Insulin-like growth factor (IGF-I) stimulates thyroid cell proliferation. Using primary cultured porcine thyroid cells, we studied the intracellular pathways that mediate the action of IGF-I on thyroid cell proliferation. IGF-I stimulates inositol phosphate accumulation, a rise in cytoplasmic free calcium [( Ca2+]i), and cell proliferation. Exposure to IGF-I results in a time- and dose-dependent accumulation of inositol monophosphate, inositol bisphosphate, and inositol trisphosphate. IGF-I also increases [Ca2+]i, measured using fura-2, a fluorescent Ca2+ indicator; the IGF-I-induced [Ca2+]i response occurs immediately, reaches a maximum within 1 min, and then slowly declines. IGF-I stimulates thyroid cell proliferation, stimulates thymidine incorporation, and increases cell numbers. The IGF-I-induced inositol phosphate accumulation and [Ca2+]i response parallel thyroid cell proliferation in a dose-dependent manner; the maximal response is observed at a concentration of 100 ng/ml IGF-I, with half-maximal stimulation at approximately 10 ng/ml. Inositol phosphate accumulation and [Ca2+]i response after IGF-I stimulation may function as intracellular messengers for thyroid cell proliferation. This report may constitute the first demonstration of IGF-I-stimulated inositol phosphate accumulation and [Ca2+]i response in the cells.

Animals↗
PubMed1988

Inhibition of inositol phosphate production is a late, Ca2+-dependent effect of D2 dopaminergic receptor activation in rat lactotroph cells.

The effect of dopamine, working through the activation of D2 receptors, on inositol phosphate production induced by thyrotropin-releasing hormone (TRH) was investigated in rat pituitary lactotroph cells. Dopamine (10 microM) did not modify the initial rapid stimulation of inositol 1,4,5-triphosphate and inositol bisphosphate observed within the first 15 s after TRH addition, but progressively inhibited the later inositol phosphate production induced by the neurohormone. This kinetics of inhibition was independent of dopamine preincubation time (from 2 to 10 min). The effect was still visible when dopamine was added after TRH. It was sensitive to pertussis toxin, was unchanged by increasing cellular cAMP levels with 8-Br-cAMP, but was greatly affected by treatments that modify the cytosolic free Ca2+ concentration. Specifically, the dopamine-induced inhibition was prevented by treatment of the cells with the Ca2+ ionophore ionomycin (100-200 nM) and was mimicked either by withdrawal of Ca2+ from the incubation medium or by blockade of voltage-gated Ca2+ channels with verapamil. The dopamine treatment did not decrease the cellular levels of the various phosphoinositides, strongly suggesting that the inhibition of inositol phosphate production is not due to precursor depletion. In isolated membranes, however, dopamine was unable to counteract the inositol phosphate accumulation triggered by TRH. Taken together, the data indicate that inhibition of inositol phosphate production is not a primary event triggered by D2 receptor activation, but is a late consequence, due to the previously demonstrated (Malgaroli, A., Vallar, L., Reza Elahi, F., Pozzan, T., Spada, A., and Meldolesi, J. (1987) J. Biol. Chem. 262, 13920-13927) inhibition by dopamine of the prolonged cytosolic free Ca2+ concentration increase induced by TRH via the activation of voltage-gated Ca2+ channels. These results are inconsistent with the possibility of a direct inhibitory coupling of D2 receptors to phospholipase C in rat pituitary lactotroph cells.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1986

Comparison of the effects of muscarine and vasopressin on inositol phospholipid metabolism in the superior cervical ganglion of the rat.

Both muscarine and vasopressin have been shown previously to increase the accumulation [3H]inositol phosphates in superior cervical ganglia in which the phospholipids were labeled with [3H] inositol. In this study, we have compared the effects of muscarine and of vasopressin on phospholipid metabolism in the ganglion. The effects of these agents on [3H]inositol phosphate accumulation are additive. The response to muscarine levels off after approximately 10 min, whereas the response to vasopressin increases for at least 30 min. The incorporation of [3H]inositol into phospholipids is enhanced in decentralized ganglia and in ganglia maintained in organ culture compared to freshly isolated ganglia. These treatments appear to potentiate the effect of muscarine on [3H]inositol phosphate accumulation, but do not affect the response of the ganglia to vasopressin. Muscarine and vasopressin also increase the incorporation of [3H]inositol into phospholipids in the ganglion. Autoradiographic techniques were used to localize the inositol-containing phospholipids in the ganglion. Muscarine increases phospholipid labeling primarily in the cell bodies of the principal ganglionic neurons, whereas vasopressin increases phospholipid labeling primarily in the neuropil. These data are consistent with the hypothesis that muscarine and vasopressin stimulate the hydrolysis of different pools of ganglionic phospholipids.

Animals↗
PubMed1986

Leukotriene B4 induces formation of inositol phosphates in rat peritoneal polymorphonuclear leukocytes.

Leukotriene B4 (LTB4) induced rapid breakdown of prelabeled inositol phospholipids in rat peritoneal polymorphonuclear leukocytes (PMNs). Formation of [3H]inositol triphosphate ([3H]IP3) was rapid, with a peak of 250-300% of the control level, after 5-15 sec of exposure to LTB4. Accumulation of [3H]inositol bisphosphate was rapid, peaking after 30 sec of treatment. Accumulation of [3H]inositol monophosphate was also rapid in the presence of LiCl. The kinetics of [3H]IP3, [3H]inositol bisphosphate, and [3H]inositol monophosphate accumulation suggest that LTB4 may interact with receptors in PMNs and activate phospholipase C which in turn induces hydrolysis of inositol-phospholipids. The agonist activities of several LTB4 analogs were employed to investigate the structure-activity relationships of LTB4 receptor-mediated activation of phosphatidylinositol hydrolysis. Increases in [3H]IP3 formation were dependent upon the concentration of LTB4 and the agonist analogs. The rank order potency of these analogs was equivalent to that of the pharmacological activity of LTB4 agonists in the PMN chemotaxis assay. Furthermore, the islet activation protein isolated from Bordetella pertussis inhibited LTB4-induced [3H]IP3 formation. The tumor-promoting phorbol myristate acetate also inhibited LTB4-induced [3H]IP3 formation. The LTB4 receptors on a partially purified PMN membrane were characterized. LTB4 binding to the receptors was stereoselective and specific. The binding affinity (Kd) of [3H] LTB4 to the receptors was 1.3 +/- 0.2 nM. The maximum density of binding was 5.5 +/- 1.8 pmol/mg of protein. The rank order potency of binding affinities of several LTB4 analogs was equivalent to that of the induction of IP3 response induced by LTB4 and analogs. These results suggest that LTB4 may interact with receptors in rat PMNs, activate G protein-regulated phospholipase C, and induce [3H]IP3 formation.

Animals↗
PubMed1987

Leukotriene-induced hydrolysis of inositol lipids in guinea pig lung: mechanism of signal transduction for leukotriene-D4 receptors.

Addition of leukotriene D4 (LTD4) to [3H]myo-inositol-labeled guinea pig lung induced rapid breakdown of inositol lipids. Formation of [3H]inositol trisphosphate was rapid, with a peak of 140-160% of the control level, 30 sec post-treatment. Formation of [3H]inositol bisphosphate and [3H]inositol monophosphate ([3H]IP1) was also rapid in the presence of LiCl. LTD4-induced [3H]IP1 formation was concentration dependent, stereoselective, and not inhibited by the cyclooxygenase inhibitor, indomethacin. Agonist analogs of LTD4 and leukotriene E4 also induced dose-dependent increases in the synthesis of [3H]IP1. The rank order potency of the agonist-induced [3H]IP1 formation was equivalent to those reported for LTD4 receptor binding, smooth muscle contraction, and thromboxane B2 biosynthesis. Furthermore, a specific receptor antagonist, SKF 102922, inhibited LTD4-induced [3H]IP1 formation in guinea pig lung. These studies suggest that LTD4 may interact with membrane receptor and activate a phospholipase C, which in turn induces the hydrolysis of inositol lipids. The hydrolysis products, diacylglycerol and inositol trisphosphate, can be regarded as the intracellular messengers for LTD4 receptors in guinea pig lung. This concept may explain a variety of pharmacological effects of leukotrienes in different types of target cells or tissues.

Animals↗
PubMed1987

An islet activating protein-sensitive G protein is involved in dopamine inhibition of angiotensin and thyrotropin-releasing hormone-stimulated inositol phosphate production in anterior pituitary cells.

In primary culture of anterior pituitary cells, dopamine inhibited the angiotensin (AII)-stimulated inositol phosphate production by 28 +/- 2.5% (n = 14), with an EC50 of 660 +/- 228 nM (n = 8). This effect was blocked by (+)-butaclamol, a specific dopamine receptor antagonist. RU 24926, a D2 specific agonist, but not SKF 38393, a specific D1 agonist, inhibited AII-stimulated inositol phosphate production, suggesting that this dopamine effect is mediated through a dopamine receptor of the D2 subtype. Dopamine also partially inhibited (25%) inositol phosphate production stimulated by thyrotropin-releasing hormone (TRH). Our results suggest that the dopamine-mediated inhibition of hormonally stimulated inositol phosphate production is probably not mediated through the known inhibitory effects of dopamine on cAMP and Ca2+ intracellular concentrations. Although unknown, the mechanism by which dopamine inhibited the AII and TRH-stimulated inositol phosphate production implicates a GTP binding protein sensitive to the islet activating protein (IAP) since dopamine effects were blocked by this toxin. The alpha subunit of the GTP binding protein involved could be one of the three ADP-ribosylated proteins found in anterior pituitary cells in primary cultures, the alpha o (39 kDa), the alpha i (41 kDa), and an alpha subunit of 40 kDa. Indeed, we show here that this 40-kDa IAP substrate, already described in a few tissues, is present in anterior pituitary cells. The negative coupling between dopamine receptors and the AII or TRH inositol phosphate production systems, could be implicated in the dopamine inhibition of the AII- and TRH-stimulated prolactin release since such an inhibition is blocked by IAP. Our results suggest that the negative regulation of inositol phosphate production is one of the mechanisms by which dopamine controls hormonally stimulated prolactin release.

Adenosine Diphosphate Ribose↗
PubMed1994

Endothelin receptor type A signals both the accumulation of inositol phosphates and the inhibition of cyclic AMP generation in rat myometrium: stimulation and desensitization.

In estradiol-dominated rat myometrium, endothelin (ET)-1 caused contraction and increased the accumulation of [3H]inositol phosphates (EC50 = 70 nM), with the sequential generation of inositol trisphosphate, inositol bisphosphate, and inositol monophosphate. There was a coincident early decrease in phosphatidyl-inositol bisphosphate. The ET-1 stimulatory effect was pertussis toxin insensitive, suggesting an activation of phospholipase C via Gq/G11 proteins. ET-1 also inhibited the generation of cAMP induced by forskolin (EC50 = 30 nM). The inhibition was maintained in Ca(2+)-depleted medium and was prevented by pertussis toxin, suggesting G(i)-mediated inhibition of adenylyl cyclase. The rank order of potency for these various ET-1 effects [ET-1 > (Thr2)-sarafotoxin-b >> ET-3], as well as the inhibitory effect displayed by BQ123, a specific ETA receptor antagonist, provided evidence for the involvement of the ETA receptor subtype. Exposure to ET-1 (15 min) resulted in concentration-dependent and homologous desensitization (40%) of the inositol phosphate response triggered by ET-1. There was virtually no recovery of ET-1-mediated inositol phosphate responses in the desensitized tissue even after 180 min of incubation. In contrast, the persistent low level of ET-1 activity that was observed in spite of several washings and in the absence of rechallenge with ET-1 was progressively revsersed and totally eliminated by BQ123. The ET-1 inhibitory effect on cAMP was also desensitized, as evidenced by the attenuation of the inhibitory effect of ET-1 after 15 min of ET-1 pretreatment. The data indicate that in rat myometrium the ETA receptor is coupled, via two distinct G proteins, to two main signal transduction cascades, which both undergo rapid desensitization.

Adenylate Cyclase Toxin↗
PubMed1994

Inositol trisphosphate and energy-force coupling in rabbit aorta.

Five-minute exposures of aortic smooth muscle to 15 microM norepinephrine (NOR) under normoxia resulted in significant increases in inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] (152%), inositol 1,4-bisphosphate [Ins(1,4)P2] (171%) and inositol 4-phosphate [Ins(4)P] (203%) contents compared with values measured in unstimulated muscle but no changes in inositol 1,3,4-trisphosphate [Ins(1,3,4)P3], inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] inositol 1/3-phosphate [Ins(1/3)P] or inositol 1,3/3,4-bisphosphate [Ins(1,3/3,4)P2] contents. Increases in Ins (1,4,5)P3 content and the contents of its by-products persisted or increased for at least 20 min of NOR exposure during normoxia. After a rapid decrease in Po2 at 5 min of NOR exposure, there were parallel decreases in Ins(1,4,5)P3 and Ins(1,4)P2 contents and in force. Ins(1,4,5)P3 and Ins(1,4)P2 contents were significantly decreased to 85 +/- 4.3% and 70 +/- 9.4% of control, respectively, at a time just after onset of relaxation. At near-maximal relaxation, Ins(1,4,5)P3 and Ins(1,4)P2 contents were decreased to 36.5 +/- 5.8% and 59.2 +/- 16.8%, respectively, of control normoxia values (P < .05). After readmission of O2 to the bubbling gas, Ins(1,4,5)P3 and Ins(1,4)P2 contents rapidly increased. Comparing decreased PO2-evoked [PCr] and [ATP] (phosphocreatine and adenosine triphosphate) decrements (measured previously; Coburn et al., 1992) with our present data, threshold [PCr] and [ATP] for a decrease in Ins(1,4,5)P3 content were shown to be 0.5 and 0.8 mM, respectively, and [PCr] and [ATP] at the time of 50% decrease in Ins(1,4,5)P3 content were 0.3 and 0.7 mM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Osmoregulatory alterations in myo-inositol uptake by bovine lens epithelial cells. Part 2: Cloning of a 626 bp cDNA portion of a Na+/myo-inositol cotransporter, an osmotic shock protein.

PURPOSE: Bovine lens epithelial cells (BLECs) accumulate osmotically active organic solutes (i.e., osmolytes) including myo-inositol when exposed to hypertonic stress (osmotic shock). In hypertonic medium, the increase in myo-inositol accumulation is attributed to an elevation in activity of Na+/myo-inositol cotransporter(s). The authors report the nature of the hypertonicity-induced enhancement of myo-inositol uptake in cultured BLECs by amplifying a 626 bp cDNA from lens cell RNA. METHODS: A portion of cDNA encoding a Na+/myo-inositol cotransporter was isolated from cultured BLECs using PCR primers designed from an established myo-inositol transporter from Madin-Darby canine kidney (MDCK) cells. Using the reverse transcription-polymerase chain reaction, a 626 bp PCR product was amplified. Its nucleic acid sequence was determined by the dideoxynucleotide method using Sequenase kit. Na+/Myo-inositol cotransporter mRNA expression in the cultured cells was demonstrated under physiological and hypertonic conditions by northern analysis of poly(A)+ RNA using the lens cell 626 bp cDNA as probe. RESULTS: The BLEC cDNA sequence was 92% identical with the Na+/myo-inositol cotransporter of MDCK cells. Myo-inositol transporter mRNA was demonstrated in cultured BLECs and was significantly induced by hypertonic stress. CONCLUSIONS: These data suggest that cultured bovine lens epithelial cell adaptation to hypertonicity involves intracellular accumulation of small organic osmolytes (i.e., myo-inositol) through elevation of myo-inositol uptake activity resulting from the upregulation of transporter mRNA.

Amino Acid Sequence↗
PubMed1993

Inositol trisphosphate-independent agonist-stimulated calcium influx in rat pancreatic acinar cells.

CCK-JMV-180 is a cholecystokinin analog that stimulates digestive enzyme secretion from pancreatic acinar cells but does not cause either generation of inositol 1,4,5-trisphosphate or depletion of the inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ storage pool. We report that CCK-JMV-180 can accelerate Ca2+ influx into fura-2-loaded dispersed rat pancreatic acini and single acinar cells. Furthermore, CCK-JMV-180 accelerates Ca2+ influx into cells microinjected with the inositol 1,4,5-trisphosphate receptor antagonist heparin and into acini loaded with the Ca(2+)-chelating agent BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid). These results indicate that agonist-stimulated Ca2+ influx can occur (a) without depletion of the inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ storage pool, (b) without a rise in cytoplasmic free Ca2+ concentrations, and (c) after blockade of inositol 1,4,5-trisphosphate receptors. They suggest that depletion of an inositol 1,4,5-trisphosphate-independent intracellular Ca2+ storage pool and/or generation of a non-inositol 1,4,5-trisphosphate second messenger by CCK-JMV-180 may be a sufficient signal for acceleration of Ca2+ influx into rat pancreatic acinar cells.

Animals↗