Studies on the properties of myo-inositol-1,4,5-trisphosphate 5-phosphatase and myo-inositol monophosphatase in bovine iris sphincter smooth muscle: effects of okadaic acid and protein phosphorylation.

In bovine iris sphincter, myo-inositol 1,4,5-trisphosphate (IP3) 5-phosphatase and myo-inositol 1-phosphate (IP1) monophosphatase are mainly localized in the microsomal and soluble fractions, respectively. Studies on the properties of these enzymes can be summarized as follows. (1) The microsomal IP3 5-phosphatase hydrolyzed IP3 to myo-inositol 1,4-bisphosphate with an apparent Km of 28 microM and Vmax of 32 nmol/min per mg protein.

Muscarinic stimulation of arachidonic acid release and prostaglandin synthesis in bovine ciliary muscle: prostaglandins induce cyclic AMP formation and muscle relaxation.

In the present study it is demonstrated that in bovine ciliary muscle, muscarinic stimulation results in: (a) release of 14C-arachidonic acid (14C-AA) and 14C-labeled prostaglandins (PGs) from muscle prelabeled with 14C-AA; (b) release of endogenous PGs, measured by means of radioimmunoassay; (c) enhanced IP3 production and (d) muscle contraction. In addition, PGs, such as PGE2 and PGD2, increased cAMP formation and induced muscle relaxation. The studies on the kinetics of 14C-AA metabolism revealed that incorporation of 14C-AA into glycerolipids and its conversion into PGs by the ciliary muscle were rapid and time-dependent.

Characterization of iris sphincter smooth muscle endothelin receptor subtypes which are coupled to cyclic AMP formation and polyphosphoinositide hydrolysis.

In the mammalian iris sphincter smooth muscle, endothelins (ET) activate both adenylate cyclase and the polyphosphoinositide cascade, and the levels of cyclic AMP (cAMP) and inositol 1,4,5-trisphosphate (IP3) produced are species specific. Radioligand binding studies, using [125I]ET-1 and [125I]ET-3 and determination of changes in cAMP, IP3 and contraction due to the peptides revealed the existence of ETA and ETB receptor subtypes in this tissue. In rabbit sphincter, ETA receptors constitute about 80% of total ET receptor population and these are coupled to IP3 production and contraction.

M3 muscarinic receptors mediate an increase in both inositol trisphosphate production and cyclic AMP formation in dog iris sphincter smooth muscle.

Pharmacological studies on pirenzepine (PZ), 4-diphenylacetoxy-N-methyl-piperidine (4-DAMP) and AFDX-116 antagonism of carbachol (CCh)-induced contraction, inositol trisphosphate (IP3) production and cAMP formation revealed the involvement of M3 receptors in these responses. The PA2 values for PZ and 4-DAMP antagonism to CCh-induced contraction were 7.1 and 9.

Protein kinase C is involved in cyclic adenosine monophosphate formation due to PGF2 alpha desensitization in bovine iris sphincter.

Purpose: To examine the mechanisms underlying the effects of PGF2 alpha receptor desensitization on agonist-induced second messenger formation and contraction in bovine iris sphincter.

Methods: Short-term PGF2 alpha receptor desensitization of the bovine iris sphincter was carried out by incubating the tissue in Krebs-Ringer bicarbonate buffer containing 25 microM PGF2 alpha for 45 min at 37 degrees C. The effects of PGF2 alpha and other pharmacologic agents on inositol 1,4,5-triphosphate (IP3) production and cyclic adenosine monophosphate (cAMP) formation in desensitized and nondesensitized tissues were monitored by anion-exchange chromatography and radioimmunoassay.

Comparative studies on fatty acid composition and phospholipases A2 and C activities in rabbit and bovine iris-ciliary body.

It is well established that production of prostaglandins by ocular tissues is dependent upon the species. The rabbit iris-ciliary body produces greater amounts of prostaglandins than that of the bovine. To throw more light on the biochemical basis underlying these differences we have compared the fatty acid composition and phospholipases A2 and C activities in rabbit and bovine irides.

Purification and characterization of phosphoinositide-specific phospholipase C from bovine iris sphincter smooth muscle.

Two forms (I and II) of phosphoinositide-specific phospholipase C (PLC) were purified from the cytosol of bovine iris sphincter by sequential chromatography on DEAE-Sepharose, EAH-Sepharose, heparin-Sepharose, Sephacryl S-200 gel filtration and Mono Q HR columns. The final step resulted in specific activities of PLC-I and PLC-II of 4.3 and 5.

Involvement of a pertussis toxin-sensitive G protein-coupled phospholipase A2 in agonist-stimulated arachidonic acid release in membranes isolated from bovine iris sphincter smooth muscle.

We have shown that in bovine iris sphincter membranes G proteins are involved in coupling muscarinic-, PGF2 alpha-, endothelin- and platelet-activating factor receptors to the activation of phospholipase A2 and the release of arachidonic acid. GTP gamma S and GTP gamma S plus carbachol stimulated arachidonic acid release in the membranes in a dose- and time-dependent manner. Nucleotide stimulation was specific to GTP gamma S, since GDP, GDP beta S and ATP had no effect.

Activation of phospholipase D by endothelin-1 and other pharmacological agents in rabbit iris sphincter smooth muscle.

The stimulation of phospholipase D (PLD) activity by endothelin-1 (ET1) was investigated in rabbit iris sphincter prelabelled with [3H]myristic acid. In the presence of 0.5% ethanol, ET1 caused a time- and dose-dependent increase in the production of [3H]phosphatidylethanol ([3H]PEt).

Carbachol stimulates adenylate cyclase and phospholipase C and muscle contraction-relaxation in a reciprocal manner in dog iris sphincter smooth muscle.

In the dog iris sphincter, muscarinic acetylcholine receptors are coupled either to the stimulation of phospholipase C and muscle contraction or to the stimulation of adenylate cyclase and muscle relaxation, this was found to be dependent upon the concentration of the muscarinic agonist. In contrast to the dog, muscarinic receptors in iris sphincters from different mammalian species were found to be coupled to phospholipase C and contraction at all concentrations of carbachol investigated (1-100 microM). In the dog sphincter, lower concentrations (less than 5 microM) of carbachol stimulated myo-inositol 1,4,5-trisphosphate (IP3) production, inhibited cAMP formation and induced contraction, and higher concentrations (greater than 5 microM) enhanced cAMP formation, inhibited IP3 production and induced relaxation.

Effects of isoproterenol and forskolin on carbachol- and fluoroaluminate-induced polyphosphoinositide hydrolysis, inositol trisphosphate production, and contraction in bovine iris sphincter smooth muscle: interaction between cAMP and IP3 second messenger systems.

We have investigated the effects of isoproterenol (ISO) and forskolin on carbachol(CCh)- and fluoroaluminate (AlF4-)-induced phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis, myo-inositol 1,4,5-trisphosphate (IP3) production, 1,2-diacylglycerol, measured as phosphatidic acid (PA) formation, and contraction in the bovine iris sphincter smooth muscle. The data from these studies can be summarized as follows. (1) CCh (20 microM) stimulated significantly PIP2 hydrolysis, IP3 production, PA formation, and contraction.

Species differences in the effects of substance P on inositol trisphosphate accumulation and cyclic AMP formation, and on contraction in isolated iris sphincter of the mammalian eye: differences in receptor density.

The effects of substance P (SP) on inositol trisphosphate (IP3) accumulation, myosin light chain (MLC) phosphorylation, cAMP formation and contraction were studied in iris sphincter smooth muscle of different mammalian species. SP receptor density was also examined in membrane fractions from this tissue. The data obtained can be summarized as follows.

The modulation of rat liver carcinogenesis by perfluorooctanoic acid, a peroxisome proliferator.

Perfluorooctanoic acid (PFOA) is a peroxisome proliferator. The aim of this study was to test for its ability to act as a positive modulator of hepatocarcinogenesis, in the so-called biphasic (initiation by diethylnitrosamine 200 mg/kg ip followed by treatment with the suspected modulators) and triphasic (initiation by the same dose of diethylnitrosamine followed by a selection procedure for 2 weeks consisting of giving 2-acetylaminofluorene and in the middle of this treatment a single dose of CCl4 followed by treatment with the suspected modulators) protocols of liver carcinogenesis. In both protocols treatment with PFOA increased the incidence of malignant hepatocellular carcinoma (HCC).

Effects of surgical sympathetic denervation on myo-inositol trisphosphate production and contraction in the dilator and sphincter smooth muscles of the rabbit iris: evidence for interaction between the cyclic AMP and calcium signaling systems.

The effects of norepinephrine (NE), carbachol (CCh), NaF, 3-isobutyl-1-methylxanthine (IBMX), and high K+ concentration (80 mM) depolarization on inositol trisphosphate (IP3) accumulation, cyclic AMP (cAMP) formation, and contraction were investigated in the dilator and sphincter smooth muscles of the sympathetically denervated as well as the normal rabbit eye. (a) In the denervated dilator muscle, NE-stimulated IP3 production and contraction are enhanced. (b) In the sphincter muscle of rabbits that have undergone sympathetic denervation.

Species differences in the effects of endothelin-1 on myo-inositol trisphosphate accumulation, cyclic AMP formation and contraction of isolated iris sphincter of rabbit and other species.

The authors investigated the effects of endothelin-1 (ET1) on inositol trisphosphate (IP3) production, 1, 2-diacylglycerol (DAG) formation, measured as phosphatidic acid (PA), cAMP formation, and contraction in iris sphincter of different mammalian species. They found that ET1 is a potent agonist for IP3 production, DAG formation, and contraction in rabbit, dog, cat, and pig iris sphincters, and for cAMP formation in all species that were investigated--rabbit, dog, cat, pig, bovine, monkey, and human sphincters. In the bovine model, ET1 induced cAMP formation in a dose-dependent manner, with an EC50 of 28 nM.

The effect of M & B 22948 on carbachol-induced inositol trisphosphate accumulation and contraction in iris sphincter smooth muscle.

The effect of a cyclic GMP phosphodiesterase inhibitor, M & B 22948, on carbachol-induced phosphatidylinositol 4,5-bis-phosphate (PIP2) breakdown and phosphatidic acid labeling, 1,4,5-inositol trisphosphate (IP3) accumulation and muscle contraction was studied in bovine iris sphincter smooth muscle. Addition of carbachol (10 microM) to 32P-labeled tissue resulted in increased labeling of phosphatidic acid and hydrolysis of PIP2. In myo[3H]inositol labeled tissue, carbachol caused rapid accumulation of IP3 which reached its maximum at about 2 min.

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.

Peroxisome proliferation and modulation of rat liver carcinogenesis by 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, perfluorooctanoic acid and nafenopin.

Using an initiation--selection--promotion protocol for induction of liver tumors in Wistar rats, the modulating action of various peroxisome proliferators on neoplasia as well as on selected biochemical parameters was studied. After treatment with diethylnitrosamine (DEN), the animals were subsequently subjected to a selection procedure involving feeding of 2-acetylaminofluorene (2-AAF), and in the middle of the 2-AAF treatment, a single necrogenic dose of carbon tetrachloride. Following a recovery period, the rats were fed a diet containing 0.

Specific binding of [3H]inositol 1,4,5-trisphosphate to bovine iris sphincter microsomal membranes.

The binding of [3H]inositol 1,4,5-trisphosphate [( 3H]IP3) to bovine iris sphincter microsomes has been shown to be rapid, reversible and saturable. The microsomal preparation contained a single population of high affinity sites for [3H]IP3 (Kd = 3.52 nM, Bmax = 218 fmol/mg protein).

Species differences in the effects of leukotriene D4 on inositol trisphosphate accumulation, cyclic AMP formation and contraction in iris sphincter of the mammalian eye.

The effects of leukotriene (LT) D4 on inositol trisphosphate (IP3) accumulation, cAMP formation, and contraction in the iris sphincter smooth muscle of different mammalian species were investigated and functional and biochemical reciprocal interactions between the IP3-Ca2+ and cAMP second messenger systems were demonstrated. The effects of the LT on the biochemical and pharmacological responses are dose- and time-dependent, and are not mediated through the release of acetylcholine or prostaglandins. Addition of LTD4 (0.

M3-muscarinic receptor subtype predominates in the bovine iris sphincter smooth muscle and ciliary processes.

The distribution of mRNAs encoding muscarinic acetylcholine receptor (mAChR) subtypes (M1, M2, M3, and M4) was investigated in the bovine iris-ciliary body by Northern blot hybridization with subtype-specific oligonucleotide probes that were complementary to unique regions of the M1, M2, M3, and M4 mAChRs. Whole rat brain RNA, which contains all four subtypes, was employed as a positive control. Both the iris sphincter and the ciliary processes were found to contain predominantly the M3 mAChR subtype and minor amounts of the M2 subtype.

Short-term desensitization of prostaglandin F2 alpha receptors increases cyclic AMP formation and reduces inositol phosphates accumulation and contraction in the bovine iris sphincter.

The effect of short-term prostaglandin (PG) desensitization on PGF2 alpha receptor-mediated inositol phosphates accumulation, 1,2-diacylglycerol production, measured as phosphatidic acid (PA), myosin light chain (MLC) phosphorylation, cAMP formation and contraction was investigated in bovine iris sphincter smooth muscle. We have found that incubation of the sphincter with 25 microM PGF2 alpha for 45 min leads to: (a) significant loss in sensitivity of the tissue to PGF2 alpha receptor-stimulated inositol phosphates accumulation, PA production, MLC phosphorylation and contraction, and (b) significant increase in both basal and PGF2 alpha-stimulated cAMP formation. These changes are probably not due to reduction in phospholipid synthesis because there were no detectable differences in basal phospholipid labeling, either from 3H-inositol or from 32P, between normal and desensitized muscles.

Activation of beta-adrenergic receptors causes stimulation of cyclic AMP, inhibition of inositol trisphosphate, and relaxation of bovine iris sphincter smooth muscle. Biochemical and functional interactions between the cyclic AMP and calcium signalling systems.

We have investigated the interactions between the cAMP and inositol 1,4,5-trisphosphate (IP3)-Ca2+ signalling systems in the bovine iris sphincter by measuring the effects of beta-adrenergic and cholinergic muscarinic agonists and antagonists on cAMP formation, IP3 accumulation and muscle contraction-relaxation. (1) Addition of 5 microM isoproterenol (ISO) or forskolin (5 microM) consistently produced stimulation of cAMP (540%), inhibition of IP3 (34%) and complete relaxation of the muscle. The ISO effects were dose-dependent, with EC50 values for cAMP formation, IP3 inhibition and muscle relaxation of 2.