Ata A. Abdel-Latif

Cross Talk Between Cyclic Nucleotides and Polyphosphoinositide Hydrolysis, Protein Kinases, and Contraction in Smooth Muscle

This article provides an update of a minireview published in 1996 (Abdel-Latif AA. Proc Soc Exp Biol Med 211:163–177, 1996), the purpose of which was to examine in nonvascular smooth muscle the biochemical and functional cross talk between the sympathetic nervous system, which governs the formation of cAMP and muscle relaxation, and the parasympathetic nervous system, which governs the generation of IP3 and diacylglycerol, from the polyphosphoinositides, Ca2+ mobilization, and contraction. This review examines further evidence, both from nonvascular and vascular smooth muscle, for cross talk between the cyclic nucleotides, cAMP and cGMP via their respective protein kinases, and the Ca2+-dependent- and Ca2+-independent-signaling pathways involved in agonist-induced contraction. These include the IP3-Ca2+-CaM- myosin light chain kinase (MLCK) pathway and the Ca2+-independent pathways, including protein kinase C-, MAP kinase-, and Rho-kinase. In addition, MLC phosphorylation and contraction can also be increased by a decrease in myosin phosphatase activity. A summary of the cross talk between the cyclic nucleotides and these signaling pathways was presented. In smooth muscle, there are several targets for cyclic nucleotide Inhibition and consequent relaxation, including the receptor, G proteins, phospholipase C-β1–4 Isoforms, IP3 receptor, Ca2+ mobilization, MLCK, MAP kinase, Rho-kinase, and myosin phosphatase. While significant progress has been made in the past four years on this cross talk, the precise mechanisms underlying the biochemical basis for the cyclic nucleotide inhibition of Ca2+ mobilization and consequently muscle contraction remain to be established. Although it is well established that second-messenger cross talk plays an important role in smooth muscle relaxation, the many sources which exist in smooth muscle for Ca2+ mobilization, coupled with the multiple signaling pathways involved in agonist-induced contraction, contribute appreciably to the difficulties found by many investigators in identifying the targets for cyclic nucleotide inhibition and consequent relaxation. Better methodology and more novel interdisciplinary approaches are required for elucidating the mechanism(s) of cAMP- and cGMP-inhibition of smooth muscle contraction.

Human ciliary muscle cell responses to FP-class prostaglandin analogs: Phosphoinositide hydrolysis, intracellular Ca2+ mobilization and MAP kinase activation

Phospholipase C induced phosphoinositide (PI) turnover, intracellular Ca(2+) ([Ca(2+)](i)) mobilization and mitogen-activated protein (MAP) kinase activation by FP-class prostaglandin analogs was studied in normal human ciliary muscle (h-CM) cells. Agonist potencies obtained in the PI turnover assays were: travoprost acid ((+)-fluprostenol; EC(50) = 2.6 +/- 0.8 nM) > bimatoprost acid (EC(50) = 3.6 +/- 1.2 nM) > (+/-)-fluprostenol (EC(50) = 4.3 +/- 1.3 nM) >> prostaglandin F(2 alpha) (PGF(2 alpha)) (EC(50) = 134 +/- 17 nM) > latanoprost acid (EC(50) = 198 +/- 83 nM) > S-1033 (EC(50) = 2930 +/- 1420 nM) > unoprostone (EC(50) = 5590 +/- 1490 nM) > bimatoprost (EC(50) = 9600 +/- 1100 nM). Agonist potencies in h-CM cells correlated well with those previously obtained for the cloned human ciliary body-derived FP receptor (r = 0.96, p< 0.001) and that present on h-TM cells (r = 0.94, p< 0.0001). Travoprost acid, PGF(2 alpha) and unoprostone also stimulated [Ca(2+)](i) mobilization in h-CM cells with travoprost acid being the most potent agonist. MAP kinase activity was stimulated in the h-CM cells with the following rank order of activity (at 100 nM): travoprost acid > PGF(2 alpha) > latanoprost acid > PGD(2) > bimatoprost > latanoprost = bimatoprost acid = fluprostenol > PGE(2) = S-1033 > unoprostone > PGI(2). The PI turnover, [Ca(2+)](i) mobilization and MAP kinase activation induced by several of these agonists was blocked by the FP receptor antagonist, AL-8810 (11 beta-fluoro-15-epiindanyl PGF(2 alpha)) (e.g. K(i) = 5.7 microM versus PI turnover). These studies have characterized the biochemical and pharmacological properties of the native FP prostaglandin receptor present on h-CM cells using three signal transduction mechanism assays and a broad panel of FP-class agonist analogs (including free acids of bimatoprost, travoprost and latanoprost) and the FP receptor antagonist, AL-8810.

Role of protein kinase C α in endothelin-1 stimulation of cytosolic phospholipase A2 and arachidonic acid release in cultured cat iris sphincter smooth muscle cells

We have investigated the role and mechanism of protein kinase C (PKC) isoforms in endothelin-1 (ET-1)-induced arachidonic acid (AA) release in cat iris sphincter smooth muscle (CISM) cells. ET-1 increased AA release in a concentration (EC50=8 nM) and time-dependent (t1/2=1.2 min) manner. Cytosolic phospholipase A2 (cPLA2), but not phospholipase C (PLC), is involved in the liberation of AA in the stimulated cells. This conclusion is supported by the findings that ET-1-induced AA release is inhibited by AACOCF3, quinacrine and manoalide, PLA2 inhibitors, but not by U-73122, a PLC inhibitor, or by RHC-80267, a diacylglycerol lipase inhibitor. A role for PKC in ET-1-induced AA release is supported by the findings that the phorbol ester, PDBu, increased AA release by 96%, that prolonged treatment of the cells with PDBu resulted in the selective down regulation of PKCα and the complete inhibition of ET-1-induced AA release, and that pretreatment of the cells with staurosporine or RO 31-8220, PKC inhibitors, blocked the ET-1-induced AA release. Go-6976, a compound that inhibits PKCα and β specifically, blocked ET-1-induced AA release in a concentration-dependent manner with an IC50 value of 8 nM. Thymeatoxin (0.1 μM), a specific activator of PKCα, β, and γ induced a 150% increase in AA release. Treatment of the cells with ET-1 caused significant translocation of PKCα, but not PKCβ, from cytosol to the particulate fraction. These results suggest that PKCα plays a critical role in ET-1-induced AA release in these cells. Immunochemical analysis revealed the presence of cPLA2, p42mapk and p44mapk in the CISM cells. The data presented are consistent with a role for PKCα, but not for p42/p44 mitogen-activated protein kinase (MAPK), in cPLA2 activation and AA release in ET-1-stimulated CISM cells since: (i) the PKC inhibitor, RO 31-8220, inhibited ET-1-induced AA release, cPLA2 phosphorylation and cPLA2 activity, but had no inhibitory effect on p42/p44 MAPK activation, (ii) genistein, a tyrosine kinase inhibitor, inhibited ET-1-stimulated MAPK activity but had no inhibitory effect on AA release in the ET-1-stimulated cells. We conclude that in CISM cells, ET-1 activates PKCα, which activates cPLA2, which liberates AA for prostaglandin synthesis.

Calcium-mobilizing receptors, polyphosphoinositides, generation of second messengers and contraction in the mammalian iris smooth muscle: historical perspectives and current status.

It is well established now that activation of Ca2+ -mobilizing receptors results in the phosphodiesteratic breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2), instead of phosphatidylinositol (PI), into myoinositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DG). There is also accumulating experimental evidence which indicates that IP3 and DG may function as second messengers, the former to mobilize Ca2+ from intracellular sites and the latter to activate protein kinase C (PKC). In this review, I have recounted our early studies, which began in 1975 with the original observation that activation of muscarinic cholinergic and adrenergic receptors in the rabbit iris smooth muscle leads to the breakdown of PIP2, instead of PI, and culminated in 1979 in the discovery that the stimulated hydrolysis of PIP2 results in the release of IP3 and DG and that this PIP2 breakdown is involved in the mechanism of smooth muscle contraction. In addition, I have summarized more recent work on the effects of carbachol, norepinephrine, substance P, the platelet-activating factor, prostaglandins, and isoproterenol on PIP2 hydrolysis, IP3 accumulation, DG formation, myosin light chain (MLC) phosphorylation, cyclic AMP production, arachidonic acid release (AA) and muscle contraction in the iris sphincter muscle. These studies suggest: (a) that the IP3-Ca2+ signalling system, through the Ca2+ -dependent MLC phosphorylation pathway, is probably the primary determinant of the phasic component of the contractile response; (b) that the DG-PKC pathway may not be directly involved in the tonic component of muscle contraction, but may play a role in the regulation of IP3 generation; (c) that there are biochemical and functional interactions between the IP3-Ca2+ and the cAMP second messenger systems, cAMP may act as regulator of muscle responses to agonists that exert their action through the IP3-Ca2+ system; and (d) that enhanced PIP2 turnover is involved in desensitization and sensitization of alpha 1-adrenergic- and muscarinic cholinergic-mediated contractions of the dilator and sphincter muscles of the iris, respectively. The contractile response is a typical Ca2+ -dependent process, which makes smooth muscle an ideal tissue to investigate the second messenger functions of IP3 and DG and their interactions with the cAMP system.

Subcellular distribution of sodium-potassium adenosine triphosphatase, acetylcholine and acetylcholinesterase in developing rat brain.

Summary The normal pattern of appearance of acetylcholinesterase, acetylcholine and sodium-potassium adenosine triphosphatase has been determined in the synaptosomal and microsomal fractions during the development of rat brain. Changes in the concentrations of acetylcholinesterase and acetylcholine were found to be gradual with development whereas that of sodium-potassium adenosine triphosphatase increased dramatically around birth and levelled off during the first 2 weeks after birth. The pattern of appearance of both enzyme systems was different in both subcellular fractions and further studies on the effect of activators and inhibitors on the two enzymes showed no relationship between their activities. The possible interrelations between the synaptosomes and microsomes were discussed.

Endothelin-1 stimulates the release of arachidonic acid and prostaglandins in rabbit iris sphincter smooth muscle: Activation of phospholipase A2

We have investigated the effects of endothelin-1 (ET1) on phospholipid hydrolysis and 3H-arachidonic acid (AA) release and prostaglandin synthesis in the rabbit iris sphincter smooth muscle. ET1 actions are concentration- and time dependent with an EC50 for AA release of 1 nM and t1/2 value of 1.5 min. We have identified the AA metabolites released by ET1, employing HPLC, as both cyclooxygenase and lipoxygenase products. The AA released by ET1 appears to derive mainly from the phosphoinositides through phospholipase A2, rather than phospholipase C activation. A key role for phospholipase A2 in AA release in the sphincter muscle is supported by the following observations. (1) Pretreatment of the labeled sphincter with the phorbol ester, PDBu (100 nM) inhibited ET1-stimulated IP3 formation, but it potentiated ET1-stimulated AA release. (2) Pretreatment of the labeled tissue with isoproterenol (5 M) inhibited ET1-stimulated IP3 production without altering AA release. (3) The potency for ET1-stimulated AA release (EC50 = 1 nM) was much higher than that for IP3 formation (EC50 = 45 nM). (4) There were considerable increases, rather than decreases, in 1, 2-diacyl-glycerol formation (1.2-folds) and its phosphorylated product, phosphatidic acid (2.6-folds) by ET1. It is concluded that in the rabbit iris sphincter ET1 is a potent agonist for AA release and eicosanoid synthesis and that AA is released from phosphoinositides mainly through activation of phospholipase A2.

In vivo incorporation of choline, glycerol and orthophosphate into lecithin and other phospholipids of subcellular fractions of rat cerebrum

1. 1. The turnover of lecithin of the membranes of synaptosomes, microsomes and 15-K fraction, isolated from rat cerebra during the stage of active myelination, has been measured by injecting intracranially [14C]choline, [14C]glycerol or [32P]orthophosphate over a period ranging from 30 min to 14 days and the average biological half-lives of lecithin from all the subcellular fractions studied for the three labeled precursors was found to be 11.7 days, 2.9 days and 18.6 days, respectively. 2. 2. The half-life of lecithin from microsomes was slightly lower than that from the other two subfractions. 3. 3. These results suggest that the metabolism of phospholipids in the nervous system is a heterogeneous process. A number of explanations for these differences in half-lives of lecithin were offered. 4. 4. All subfractions studied were found to incorporate the three labeled precursors into their respective major phospholipids. 5. 5. The rate of incorporation of the radioactive precursors into lecithin of the various subcellular fractions was in the following order : microsomes > 15-K > synaptosomes. 6. 6. These data suggest that at least part of the phospholipids can be synthesized at the synapse and this synthesis is probably independent of the cell body of the neuron.

Metabolism of Phosphoinositides

The literature on the metabolism of phosphoinositides, phosphatidylinositol (PI),* and the polyphosphoinositides, phosphatidylinositol-4-phosphate (PI-P; diphosphoinositide, DPI), and phosphatidylinositol-4,5-bisphosphate (Pl-bis P; triphosphoinositide, TPI), prior to 1970 was reviewed by Hawthorne and Kai1 in the earlier edition of the Handbook of Neurochemistry. Therefore, in this chapter, I limit my discussion to work that has been published since then, giving background summaries of earlier studies only when necessary to complete the overall picture.

Studies on the properties of triphosphoinositide phosphomonoesterase and phosphodiesterase of rabbit iris smooth muscle

The rabbit iris smooth muscle has been shown to contain triphosphoinositide phosphomonoesterase (phosphatidyl-myo-inositol-4,5-bisphosphate phosphohydrolase, EC 3.1.3.36) and phosphodiesterase (triphosphoinositide inositoltrisphosphohydrolase, EC 3.1.4.11) activities. Under our experimental conditions about 77% of the phosphomonoesterase and 61% of the phosphodiesterase activities were localized in the particulate fraction. The kinetic properties of the enzymes in the microsomal fraction were examined. The enzyme preparation was specific to polyphosphoinositides; it did not attack phosphatidylinositol under the present assay condition. The effects of Ca2+ and Mg2+ were also studied. Although the microsomal enzymes did not require added divalent cations for their activities, both the phosphomonoesterase and phosphodiesterase were appreciably inhibited by 1 mM EDTA. Phosphodiesterase and phosphomonoesterase were stimulated by Ca2+ and Mg2+, respectively. The demonstration of triphosphoinositide phosphodiesterase in the iris muscle, coupled with the findings that this enzyme is activated by Ca2+ and is not influenced by acetylcholine add further support to our previous conclusion (J. Pharmacol. Exp. Ther. (1978) 204, 655--668; J. Neurochem. (1978) 30, 517--525) that an increased Ca2+ influx, following the interaction between the neurotransmitter and its receptor, could act to stimulate the phosphodiesterase, thus leading to increased triphosphoinositide breakdown and increased phosphatidic acid via increased diacylglycerol.

Effects of dl-propranolol on the synthesis of glycerolipids by rabbit iris muscle

Abstract Propranolol (0.03−0.3 mM), an amphiphilic cationic drug which is used therapeutically as a β-blocker, was found to alter significantly the incorporation of [ 14 C]glucose, [ 14 C]glycerol, [ 14 C]acetate, 32 Pi, [ 3 H]cytidine, [ 3 H]inositol, [ 14 C]choline, [ 14 C]ethanolamine and [ 14 C]serine into phospholipids of the iris muscle. Furthermore, it was found to exert a stimulatory effect on the [ 14 C]serine incorporation into phosphatidylserine of the muscle and microsomes. In contrast, sotalol, another β-blocker-but lacking the hydrophobicity of propranolol-exerted no effect on lipid metabolism. Whereas norepinephrine stimulated only the turnover of the phosphate moiety of phosphatidic acid and phosphatidylinositol, in general propranolol caused the following changes: (a) it stimulated by 2- to 6-fold the labelling of phosphatidic acid and phosphatidylinositol from [ 14 C]glucose, [ 14 C]glycerol, [ 14 C]acetate, 32 Pi and [ 3 H]inositol, (b) it increased by 5- and 38-fold the incorporation of 32 Pi and [ 3 H]cytidine, respectively into CDP-diglyceride, (c) it inhibited appreciably the incorporation of [ 14 C]glucose, [ 14 C]glycerol, [ 14 C]acetate and 32 Pi into phosphatidylcholine and phosphatidylethanoalmine. However, while it inhibited significantly the [ 14 C]choline incorporation into the former, it stimulated by 60 per cent the ethanolamine incorporation into the latter phospholipid. These results indicate that propranolol probably redirects phospholipid synthesis de novo , by inhibiting phosphatidate phosphohydrolase, such that the increase obtained in the biosynthesis of phosphatidylinositol is accompanied by a corresponding decrease in the synthesis of phosphatidylcholine and phosphatidylethanolamine. Propranolol also caused a 250 per cent increase in the [ 14 C]serine incorporation into phosphatidylserine of the iris muscle and 28 per cent increase in that of microsomes. The drug appears to stimulate the Ca 2+ -uptake by muscle and microsomes, which in turn could act to stimulate the Ca 2+ -catalyzed base-exchange reaction. In addition the metabolic pathways involved in the biosynthesis of the major phospholipids of the iris, a smooth muscle, are reported for the first time. These pathways were found to be essentially similar to those reported for other tissues.