Pain management during diagnostic office hysteroscopy in postmenopausal women: a randomized study.

Objective: This study aimed to compare efficacy and safety of lidocaine versus tramadol versus placebo in reducing the pain of diagnostic outpatient hysteroscopy (OH) in postmenopausal women.Materials and methods: This randomized double-blinded study included 156 menopausal women who received intrauterine lidocaine infusion or oral tramadol (50 mg) or placebo before diagnostic OH (52 women/group). Primary outcome was pain severity during the procedure using a 10-cm visual analog scale. Secondary outcomes were pain scores 10 and 30 min post procedure, satisfaction level, and ease of cervical entry.Results: Lidocaine had lower pain scores compared to placebo during and 10 min after the procedure (p < 0.001). Tramadol had lower pain scores than placebo during the procedure (p = 0.04), 10 min after the procedure (p = 0.03), and 30 min after the procedure (p = 0.04). Both lidocaine and tramadol resulted in an easier procedure than placebo (p < 0.001 and p = 0.04, respectively). Lidocaine had an easier cervical entry compared to tramadol (p = 0.004). Satisfaction scores in the lidocaine and tramadol groups were significantly higher than in the placebo group (p < 0.001).Conclusions: Lidocaine and tramadol were effective in reducing postmenopausal women-reported pain during and after diagnostic OH. However, lidocaine was better than tramadol in facilitating hysteroscope passage through the cervical canal and the reduction in pain levels with lidocaine was clinically relevant.Trial registration number: NCT03701984.

Neurobehavioral and Menstrual Disorders among Adolescent Females Environmentally Exposed to Pesticides, Menoufia Governorate, Egypt.

BACKGROUND: Adolescent females living in agricultural areas where crops are routinely sprayed by pesticides are expected to be environmentally exposed to pesticides' health hazards partially as those occupationally exposed. OBJECTIVE: to assess menstrual and neurobehavioral disorders among adolescent females environmentally exposed to pesticides. METHODS: This was a cross-sectional study conducted on 100 pesticide exposed adolescent females who had one or more of family members are pesticides' seasonal applicators and 50 non- exposed adolescent females matched for age and education, served as controls at Menoufia governorate, Egypt during the period of pesticide application season of cotton crop from the first days of May to the end of September 2017. A self-administered and a series of neurobehavioral tests were administered and serum Acetylcholinesterase (AChE) activity was assessed. RESULTS: A significant lower AChE activity levels were found in the exposed group than controls (Mean±SD=238.49± 23.83 vs 303.35±78.54 IU/L; respectively). There were significant higher mean scores of trail making test (parts 1 and 2) and significant lower mean scores of (similarities test, Benton visual retention test, block design test, Santa Ana dexterity test (dominant and non-dominant hands) and Beery visuo-motor imitation test in the exposed group than the controls (P<0.05). Also, the exposed group reported more prevalent irregular menstrual cycle (26.8%) and intermenstrual bleeding (28.2%) compared to the control participants (8.1% and 8.1%; respectively). CONCLUSION: Adolescent females living in agricultural areas and from families whose one or more members are pesticides' applicators have significantly lower neurobehavioral performance, report more prevalent menstrual irregularities and have lower levels of serum AChE compared to a control group. The neurobehavioral deficits demonstrated a dose-response relationship AChE levels in the exposed participants. This necessitates the need for implementation of health education programs to prevent or reduce health effects associated with pesticide exposure to adolescent females.

Sodium ion and the neutrotransmitter-stimulated 32P labelling of phosphoinositides and other phospholipids in the iris muscle.

The effects of Na+, other cations and the neurotransmitters, acetylcholine and norepinephrine on 32Pi incorporation into phospholipids of the rabbit iris smooth muscle were investigated [1]. The basal 32P-labelling of phospholipids including phosphatidic acid, phosphatidylinositol, phosphatidylcholine, phosphatidylethanolamine and the polyphosphoinositides increased with Na+ concentration [2]. The neurotransmitter-stimulated 32P labelling of phosphatidic acid, phosphatidylinositol and phosphatidylcholine is dependent on the presence of extracellular Na+ [3]. The monovalent cation requirement for Na+ specific. Of the monovalent cations Li+, NH+4, K+, Choline+ and Tris, only Li+ partially substituted for Na+ [4]. A significant decrease in 32P labelling of phospholipids in response to acetylcholine was observed when Ca2+ and/or K+ were added to an isoosmotic medium deficient of Na+ [5]. Ouabain, which blocks the Na+-pump, inhibited the basal 32Pi incorporation into phosphatidylcholine and the acetylcholine-stimulated 32P labelling of phosphatidic acid, phosphatidylinositol and phosphatidylcholine [6]. It was suggested that phosphoinositide breakdown is associated with Ca2+ influx as we have previously reported (Akhtar, R.A. and Abdel-Latif, A.A. (1978) J. Pharmacol. Exp. Ther. 204, 655-668) and that the enhanced 32P-labelling of phosphoinositides could be associated with Na+ outflux, via the Na+-pump mechanism.

Role of protein kinase C alpha 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 PKCalpha 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. Gö-6976, a compound that inhibits PKCalpha and beta specifically, blocked ET-1-induced AA release in a concentration-dependent manner with an IC50 value of 8 nM. Thymeatoxin (0.1 microM), a specific activator of PKCalpha, beta, and gamma induced a 150% increase in AA release. Treatment of the cells with ET-1 caused significant translocation of PKCalpha, but not PKCbeta, from cytosol to the particulate fraction. These results suggest that PKCalpha 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 PKCalpha, 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 PKCalpha, which activates cPLA2, which liberates AA for prostaglandin synthesis.

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.

Studies on the incorporation of [1-14C]arachidonic acid into glycerolipids and its conversion into prostaglandins by rabbit iris. Effects of anti-inflammatory drugs and phospholipase A2 inhibitors.

The effects of the anti-inflammatory drugs, indomethacin and aspirin, and the phospholipase A2 inhibitors, p-bromophenacyl bromide and mepacrine, on the in vitro metabolism of [1-14C]arachidonic acid by rabbit iris smooth muscle and iris microsomes were investigated. The incorporation of arachidonate into glycerolipids and its conversion into prostaglandins were rapid and time-dependent. About 65% of the total radioactivity was recovered in triacylglycerol, followed by that in phosphatidylcholine (20%), diacylglycerol (6%), phosphatidylethanolamine (5%) and phosphatidylinositol (3%), respectively. Time-course studies on arachidonate release from glycerolipids of prelabelled tissue showed that triacylglycerol, phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol are the major source for arachidonate in prostaglandin synthesis in this tissue. Arachidonate release from glycerolipids was not blocked by indomethacin and the effects of the phospholipase A2 inhibitors were nonspecific. p-Bromophenacyl bromide inhibited the labelling of glycerolipids in a dose-dependent manner. Mepacrine stimulated the labelling of phosphatidic acid, phosphatidylinositol and diacylglycerol, and inhibited that of phosphatidylcholine, phosphatidylethanolamine and triacylglycerol. At concentrations under 0.25 mM it stimulated prostaglandin synthesis in microsomes and at concentrations over 0.25 mM it inhibited their synthesis in both muscle and microsomes. Indomethacin and aspirin moderately increased the labelling of glycerolipids; however, both drugs inhibited prostaglandin synthesis by iris and iris microsomes in a dose-dependent manner. Possible explanations for mechanisms underlying these effects were presented. It is concluded that the phospholipase A2 inhibitors and the anti-inflammatory drugs exert profound effects on the incorporation of [1-14C]arachidonate into glycerolipids of the rabbit iris and on its conversion into prostaglandins by both iris and iris microsomes.

Studies on the properties of a soluble phosphatidylinositol-phosphodiesterase of rabbit iris smooth muscle.

Some properties of the soluble phosphatidylinositol phosphodiesterase (monophosphatidylinositol inositolphosphohydrolase, EC 3.1.4.10) of rabbit iris smooth muscle are described. Studies on its subcellular distribution showed that in this tissue the phosphodiesterase is not exclusively cytosolic. Thus, under our experimental conditions about 58% of the enzyme activity was found in the soluble fraction and the remainder was particulate. When the latter was treated with deoxycholate about 59% of the enzyme activity, compared to 86% of that of ATPase, was still bound to the particulate fraction. The kinetic properties of the enzyme (30--50% (NH4)2SO4 fraction) were examined. Maximum breakdown was 7.7 mumol/h per mg protein and occurred at pH 5.6. The products of [14C]arachidonic acid-labelled phosphatidylinositol were 1,2-diacylglycerol and a mixture of 86% myoinositol 1-phosphate and 14% myoinositol 1,2-(cyclic)phosphate. The enzyme has an absolute requirement for Ca2+. Addition of Ba2+, La3+, Mg2+, Mn2+, EGTA or EDTA at 0.05--5 mM concentrations; Sr2+ at higher concentrations (greater than 0.25 mM) markedly inhibited the phosphodiesterase activity and this inhibition was completely reversed by Ca2+. The enzyme is specific for the phosphoinositides.

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. The IP1 monophosphatase in the soluble fraction hydrolyzed IP1 into free inositol with an apparent Km of 89 microM and Vmax of 7 nmol/min per mg protein. (2) IP3 5-phosphatase and IP1 monophosphatase had optimal pH values at 8.0 and 7.0, respectively. (3) Both enzymes required Mg2+ and their highest specific activities were at a cation concentration of 2 mM. (4) Ca2+ (> 0.5 microM) exerted an inhibitory effect on IP3 5-phosphatase activity, and marked inhibition (47%) was observed at a concentration of 10 microM. Higher concentrations of the cation (> 100 microM) were required to inhibit IP1 monophosphatase. (5) IP1 monophosphatase, but not IP3 5-phosphatase, was inhibited by Li+. Li+ had no effect on the contractile response in this smooth muscle. (6) Both enzymes were inhibited by ATP and by the thiol-blocking agent, disulfiram. In addition, thimerosal, a thiol reagent, also inhibited the IP3 5-phosphatase activity. (7) Protein phosphorylation of the microsomal and soluble fractions with PKA or PKC had no effect on the activities of these enzymes. (8) Okadaic acid, a protein phosphatase inhibitor, had no effect on the activity of IP3 5-phosphatase. However, in the intact iris sphincter the toxin significantly reduced the carbachol-induced IP3 production, 1,2-diacylglycerol formation, measured as phosphatidic acid, and caused muscle relaxation.

Species differences in the effects of prostanoids on MAP kinase phosphorylation, myosin light chain phosphorylation and contraction in bovine and cat iris sphincter smooth muscle.

There is evidence from our own laboratory and that of others that EP-receptor ligands are strong contractile agonists in bovine iris sphincter and that FP-receptor agonists are strong contractile agonists in cat iris sphincter. Here, we have investigated the effects of prostaglandin (PG) receptor agonists of the FP-, EP-, TP- and DP-class on myosin light chain (MLC) phosphorylation, p42/p44 MAP kinase phosphorylation and contraction in the iris sphincter of bovine and cat. Using three signal transduction mechanism assays, namely MLC phosphorylation, MAP kinase phosphorylation and contraction, we demonstrated that in bovine iris sphincter the rank order of potency of the PG agonists in the contractile and MLC phosphorylation assays is as follows: E2>U46619>F2alpha>D2, and in cat F2alpha>D2>E2>U46619. In the MAP kinase assay, in bovine iris sphincter the rank order of potency is E2>F2alpha and in cat F2alpha>E2. These conclusions are supported by the following findings: (1) In the contractile assay, in the bovine sphincter the EC50s for PGF2alpha, PGE2, U46619 and PGD2 were found to be 1.4x10(-7), 5.0x10(-9), 9.0x10(-9) and 1.3x10(-6)M, respectively, and the corresponding values in the cat were 1.9x10(-8), 2.3x10(-7), 1.5x10(-6) and 6.9x10(-8)M, respectively. (2) In the MLC phophorylation assay, in the bovine sphincter PGF2alpha, PGE2, U46619 and PGD2 increased MLC phophorylation by 118%, 165%, 153% and 72%, respectively, and the corresponding values in cat were 175%, 99%, 90% and 95%, respectively. (3) In the MAP kinase assay, in the bovine iris sphincter PGF2alpha and PGE2, increased MAP kinase phosphorylation by 276% and 328%, respectively, and the corresponding values in cat were 308% and 245%, respectively. The data presented demonstrate pronounced species differences in the effects of the prostanoids on the MLC kinase signaling pathway in bovine and cat irides and furthermore confirm the existence of FP-receptors in that of the bovine.

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). Within 30 s the peptide increased PEt formation by 30% and after 5 min increased it by 140%. The EC50 value for ET1-stimulated PEt formation was found to be 30 nM. This value is appreciably lower than the EC50 we previously obtained for ET1-induced inositol trisphosphate production (45 nM), but considerably higher than that for arachidonic acid release (1 nM). PEt formation was significantly stimulated by prostaglandin F20, phorbol 12,13-dibutyrate (PDBu), chloroform, A23187 and A1F4-, but it was not affected by carbachol or the platelet-activating factor. PDBu-stimulated PEt formation was blocked by staurosporine and it was not potentiated by A23187. Staurosporine had no effect on ET1-stimulated PEt formation. Our data indicate that ET1 stimulation of PLD occurs independently of protein kinase C activation, phospholipase C activation and intracellular Ca2+ mobilization, and phospholipase A2 activation. In this tissue the ET1 receptor is probably coupled to the three phospholipases through several G-proteins, and this appears to be species and receptor type specific.

Activation of particulate guanylate cyclase by adrenomedullin in cultured SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells.

We investigated the effects of adrenomedullin (ADM) on cGMP production in cultured SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells. ADM increased cGMP accumulation in a time- and concentration- dependent manner. The peptide increased cGMP formation in the transformed cells by 405-fold as compared to 1. 6-fold in primary cultured CISM cells. The basal cGMP concentrations in both cell types were comparable. In addition, ADM increased cAMP accumulation in SV-CISM-2 cells and in primary cultured cells by 18. 9- and 5.8-fold, respectively. The ADM receptor antagonist, ADM(26-52), but not the atrial natriuretic peptide (ANP) receptor antagonist, anantin, inhibited ADM-induced cGMP formation. The phorbol ester, phorbol 12, 13-dibutyrate (PDBu), which inhibits particulate guanylate cyclases in smooth muscle, blocked ADM-stimulated cGMP accumulation. In contrast, inhibitors of the soluble guanylate cyclases, such as LY83583 and ODQ, and inhibitors of the nitric oxide cascade had little effect on ADM-stimulated cGMP production. The stimulatory effect of ADM on cGMP formation is due to activation of the guanylate cyclase system and not to a much reduced phosphodiesterase activity. ADM stimulated guanylate cyclase activity in membrane fractions isolated from SV-CISM-2 cells in a concentration-dependent manner with EC(50) value of 72 nM. Pertussis toxin, an activator of the G-protein, Gi, inhibited ADM-stimulated cGMP accumulation, whereas cholera toxin, a stimulator of the Gs G-protein and subsequently cAMP accumulation, had little effect. Pretreatment of the plasma membrane fraction with Gialpha antibody attenuated ADM-stimulated guanylate cyclase activity by 75%. We conclude that ADM increases intracellular cGMP levels in SV-CISM-2 cells through activation of the ADM receptor and subsequent stimulation of a Gi-mediated membrane-bound guanylate cyclase.

Atrial natriuretic peptide provokes a dramatic increase in cyclic GMP formation and markedly inhibits muscarinic-stimulated Ca2+ mobilisation in SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells.

We investigated the effects of cGMP-elevating agents, including atrial natriuretic peptide (ANP), C-type natriuretic peptide (CNP) and sodium nitroprusside (SNP), on cGMP accumulation and on carbachol (CCh)-stimulated intracellular calcium ([Ca2+]i) mobilisation in SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells and in primary cultured cat iris sphincter smooth muscle (CISM) cells. The stimulatory effects of the natriuretic peptides on cGMP production correlated well with their inhibitory effects on CCh-induced [Ca+1]i mobilisation, and these effects were significantly more pronounced in the SV-CISM-2 cells than in the CISM cells. Thus, ANP (1 microM) increased cGMP production in the SV-CISM-2 cells and CISM cells by 487- and 1.7-fold, respectively, and inhibited CCh-induced [Ca2+]i mobilisation by 95 and 3%, respectively. In the SV-CISM-2 cells, ANP and CNP dose dependently inhibited CCh-induced [Ca2+]i mobilisation with IC50 values of 156 and 412 nM, respectively, and dose dependently stimulated cGMP formation with EC50 values of 24 and 88 nM, respectively, suggesting that the inhibitory actions of the peptides are mediated through cGMP. Both ANP and CNP stimulated cGMP accumulation in a time-dependent manner. The potency of the cGMP-elevating agents were in the following order: ANP>>CNP>>SNP; these agents had no effect on cAMP accumulation. The inhibitory effects of the natriuretic peptides were mimicked by 8-Br-cGMP, a selective activator of cGMP-dependent protein kinase. LY83583, a soluble guanylyl cyclase inhibitor, significantly inhibited SNP-induced cGMP formation but had no effect on those of ANP and CNP. The basal activities of the guanylyl cyclase and the dissociation constant (Kd) and total receptor density (Bmax) values of the natriuretic peptide receptor for [125I]ANP binding were not significantly different between the two cell types. The cGMP system, as with the cAMP system, has a major inhibitory influence on the muscarinic responses in the iris sphincter smooth muscle cells, and SV-CISM-2 cells can serve as an excellent model for investigating the cross talk between cGMP and the Ca2+ signalling system.

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. (2) Addition of ISO (0.1-25 microM), which raises the tissue cAMP level, to muscle precontracted with CCh attenuated PIP2 hydrolysis, IP3 production, PA formation and contraction in a time- and dose-dependent manner. (3) AlF4- (10 microM) induced a slow but progressive hydrolysis of PIP2, accompanied by parallel production of IP3, formation of PA, and contraction of the smooth muscle. The effects of AlF4- were dose-dependent and inhibited by deferoxamine, an Al3+ ion chelator. (4) Both forskolin (1-25 microM), which directly stimulates adenylate cyclase, and ISO inhibited the responses induced by AlF4- (10 microM) in a dose-dependent manner. (5) NaF (1-5 mM) had no effect on the activity of phospholipase C (PLC), purified from bovine iris sphincter. Furthermore, phosphorylation of the enzyme by catalytic subunit of protein kinase A had no inhibitory effect on PLC activity against PIP2. In conclusion, neither the muscarinic receptor nor PLC are the target sites for cAMP inhibition; instead the putative G-protein, which couples the activated muscarinic receptor to PLC, may be phosphorylated by cAMP-dependent protein kinase. This could attenuate the stimulation of PLC by the G-protein, thus resulting in inhibition of PIP2 hydrolysis and consequently leading to muscle relaxation. These results demonstrate cross-talk between the cAMP and IP3-Ca2+ second messenger systems and suggest that this could constitute a regulatory mechanism for the process of contraction-relaxation in smooth muscle.

Inhibition of muscarinic-stimulated polyphosphoinositide hydrolysis and Ca2+ mobilization in cat iris sphincter smooth muscle cells by cAMP-elevating agents.

The effects of carbachol (CCh) on inositol 1,4,5-trisphosphate (IP3) production and intracellular calcium ([Ca2+]i) mobilization, and their regulation by cAMP-elevating agents were investigated in SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells. CCh produced time- and dose-dependent increases in IP3 production; the t1/2 and EC50 values were 68 s and 0.5 microM, respectively. The muscarinic agonist provoked a transient increase in [Ca2+]i which reached maximum within 77 s, and increased [Ca2+]i mobilization in a concentration-dependent manner with an EC50 of 1.4 microM. Thapsigargin, a Ca(2+)-pump inhibitor, caused a rapid rise in [Ca2+]i and subsequent addition of CCh was without effect. Both CCh-induced IP3 production and CCh-induced [Ca2+]i mobilization were more potently antagonized by 4-DAMP, an M3 muscarinic receptor antagonist, than by pirenzepine, an M1 receptor antagonist, suggesting that both responses are mediated through the M3 receptor subtype. Treatment of the cells with U73122, a phospholipase C (PLC) inhibitor, resulted in a concentration-dependent decrease in both CCh-stimulated IP3 production and [Ca2+]i mobilization. These data indicate close correlation between enhanced IP3 production and [Ca2+]i mobilization in these smooth muscle cells and suggest that the CCh-stimulated increase in [Ca2+]i could be mediated through increased IP3 production. Isoproterenol (ISO) inhibited CCh-induced IP3 production (IC50 = 80 nM) and [Ca2+]i mobilization (IC50 = 0.17 microM) in a concentration-dependent manner. Microsomal fractions isolated from SV-CISM-2 cells contained phospholipase C (PLC) which was stimulated by CCh (10 microM) and GTP gamma S (0.1 microM). Pretreatment of the cells with ISO or forskolin, 5 microM each, produced membrane fractions in which CCh-stimulated PLC activity was significantly attenuated. Furthermore, when microsomal fractions isolated from SV-CISM-2 cells were phosphorylated with Protein kinase A (PKA), the CCh- and GTP gamma S-stimulated IP3 production were significantly inhibited. It can be concluded from these studies that in SV-CISM-2 cells, activation of M3 muscarinic receptors results in stimulation of PLC-mediated PIP2 hydrolysis, generating IP3 which mobilizes [Ca2+]i. Furthermore, elevation of cAMP may inhibit IP3 production and [Ca2+]i mobilization through mechanisms involving PKA-dependent phosphorylation of PLC, G-proteins, IP3 receptor and/or IP3 metabolizing enzymes.

Phorbol ester-induced protein phosphorylation and contraction in sphincter smooth muscle of rabbit iris.

Phorbol 12,13-dibutyrate (PDBu) was employed in studies designed to determine the role of C-kinase in muscle contraction in the iris sphincter. PDBu induced MLC phosphorylation and contraction in a dose- and time-dependent manner. Maximum responses exerted by PDBu were about 50-60% of that obtained with CCh, and were totally dependent on the presence of extracellular Ca2+. PDBu had no effect on basal IP3 levels, however it blocked the CCh-stimulated accumulation of IP3. PDBu-induced effects were potentiated by ionomycin, and inhibited by the C-kinase antagonist H-7. These results provide further evidence for the involvement of C-kinase in mediating the sustained phase of the contractile response in the iris sphincter.

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. This is the first report that showed an effect of the peptide on the adenylate cyclase system. In rabbit sphincter, ET1 induced a significant increase in IP3 production by 30 sec and reached a 6-fold level more than control within 1 and 5 min. ET1-stimulated IP3 production is dose dependent with an EC50 of 45 nM, this value is about 100- and 56-fold lower than those we reported for substance P and carbachol, respectively. ET1 also increased 32P labeling of PA more than 6-fold; and in rabbit sphincter, ET1 is a more potent agonist in contracting the sphincter than in contracting the dilator (the EC50 values for sphincter and dilator were 46 and 120 nM, respectively). L-type Ca2+ channels are not involved in IP3- and contraction responses because several blockers of these channels did not affect the ET1-induced responses, implying that in the iris sphincter, ET1 elicits the physiologic response through the G protein activation of phospholipase C and/or adenylate cyclase and not through the activation of voltage-dependent Ca2+ channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Alpha 1-adrenergic receptor induced subsensitivity and supersensitivity in rabbit iris-ciliary body. Effects on myo-inositol trisphosphate accumulation, arachidonate release, and prostaglandin synthesis.

Topical treatment of the rabbit eye with three successive doses of 2% epinephrine resulted in attenuation of the in vitro drug response of the alpha 1-adrenoceptor-mediated phosphoinositide hydrolysis system and of alpha 1-adrenergic receptor-mediated contraction in the iris. In contrast, sympathetic denervation of the eye resulted in potentiation of these responses. Desensitized tissues showed a significant decrease in epinephrine-induced myo-inositol trisphosphate (IP3) accumulation, 1,2-diacylglycerol (DG) formation, measured as phosphatidate, arachidonic acid (AA) liberation, measured by radiochromatography, prostaglandin (PG) E2 release, measured by radiochromatography and radioimmunoassay, and muscle contraction. Adrenergic desensitization of the eye resulted in attenuation of: The polyphosphoinositide response in the iris, measured both as loss of 32P-radioactivity from phosphatidylinositol 4,5-bisphosphate (PIP2) and as IP3 accumulation; the epinephrine-stimulated liberation of AA, from membrane phosphoinositides and other phospholipids, and PGE2 release in the iris; and the epinephrine-induced muscle contraction in the iris dilator. This adrenergic desensitization of the eye is reversible. Surgical sympathetic denervation, previously found to increase alpha 1-adrenoceptor mediated accumulation of IP3 and contraction, increased AA liberation. Dexamethasone blocked the epinephrine-induced liberation of AA and PG release, both in vivo and in vitro. These data support the hypothesis that changes in the activity of the alpha 1-adrenergic receptor-mediated phosphoinositide hydrolysis system and its derived second messengers may underlie the mechanism of adrenergic subsensitivity and supersensitivity in the iris-ciliary body. How much the desensitization of alpha 1-adrenergic receptor-mediated responses contribute to the therapeutic action of epinephrine in the eye remains to be determined.

Actions of C-type natriuretic peptide and sodium nitroprusside on carbachol-stimulated inositol phosphate formation and contraction in ciliary and iris sphincter smooth muscles.

PURPOSE: To investigate the effects of C-type natriuretic peptide (CNP) and sodium nitroprusside (SNP) on cyclic guanosine monophosphate (cGMP) accumulation and on carbachol (CCh)-stimulated inositol 1,4,5-triphosphate (IP3) production and contraction in ciliary muscle (CM) and iris sphincter (Sph) isolated from bovine and other mammalian species. METHODS: Ciliary muscle and sphincter isolated from cows, cats, dogs, rabbits, monkeys, and humans were used. Bovine specimens were used in the present work. Accumulation of cGMP and cyclic adenosine monophosphate (cAMP) in tissue extracts was measured by radioimmunoassay, IP3 production was measured by ion-exchange chromatography, and changes in tension were recorded isometrically. RESULTS: In general, CNP and SNP exerted differential inhibitory effects on muscarinic-receptor-induced responses in CM and Sph isolated from the various species. Thus in bovine CM, SNP stimulated cGMP formation in a time- and concentration-dependent manner and dose dependently inhibited CCh-induced IP3 production and contraction. These effects were inhibited by LY 83583, a soluble guanylyl cyclase inhibitor, and mimicked by 8-Br-cGMP, a cell-membrane permeable analogue of cGMP. The inhibitory effects of the soluble cGMP analogue are tissue and species specific. Sodium nitroprusside had no effect on the muscarinic responses in bovine Sph, but it attenuated CCh-induced contractions in Sph isolated from cats, dogs, and rabbits. In bovine Sph, CNP increased cGMP accumulation in a time- and dose-dependent manner and dose dependently inhibited CCh-induced IP3 production and contraction. LY 83583 had no effect on the muscarinic responses. C-type natriuretic peptide attenuated CCh-induced contraction in CM isolated from monkey and human, but it had no influence on this response in CM isolated from cows, cats, and dogs. CONCLUSIONS: In bovine CM, SNP effects are probably mediated through soluble guanylyl cyclase, whereas in Sph the CNP effects are mediated through membrane-bound guanylyl cyclase, which is associated with the type-B natriuretic peptide receptor. Agents that strongly increase intracellular cGMP levels, including SNP and CNP, produce significant inhibition of CCh-induced IP3 production and contraction. These effects are tissue and species specific. The results indicate that the cGMP signaling system, similar to the cAMP system, has a major inhibitory influence on the muscarinic responses in smooth muscles of the iris-ciliary body. The agents CNP and SNP, which stimulate cGMP accumulation in the ocular smooth muscles, could reduce intraocular pressure, presumably by increasing uveoscleral outflow induced by relaxation of the CM. However, the relationships between the CNP- and SNP-induced inhibition of the muscarinic stimulation and the reported intraocular pressure-lowering effects of the cGMP-elevating agents remain to be determined.

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. RESULTS: In the isolated bovine iris sphincter, protein kinase C (PKC) is involved in the activation of adenylate cyclase and the desensitization of prostaglandin F2 alpha receptor-mediated responses supported by these findings. (A) Exposure of the tissue to phorbol 12,13-dibutyrate, used to activate PKC, enhanced basal cAMP formation in a dose (EC50 = 8.8 x 10(-8) M) and time (t1/2 = 7.5 min) dependent manner. Phorbol 12,13-dibutyrate increased cAMP levels by twofold and it potentiated the isoproterenol-induced cAMP formation. The biologically inactive phorbol ester, 4 alpha-phorbol had no effect. Staurosporine, a potent PKC inhibitor, inhibited phorbol 12,13-dibutyrate-induced cAMP formation in a dose-dependent manner (IC50 of 0.25 microM). The increase in cAMP levels by phorbol 12,13-dibutyrate results from stimulation of adenylate cyclase, rather than from inhibition of cAMP phosphodiesterase, and it is not mediated through Ca2+ mobilization. Pretreatment of the tissue with phorbol 12,13-dibutyrate inhibited IP3 production in response to PGF2 alpha. (B) Desensitization of the sphincter with PGF2 alpha for 45 min increased cAMP formation and attenuated IP3 production and contraction. The effects of PGF2 alpha desensitization were reversed by pretreatment of the tissue with staurosporine. Down-regulation of PKC prevented the PGF2 alpha-stimulated increase in cAMP formation. In the desensitized tissue, diacylglycerol, the endogenous activator of PKC, may arise from phosphatidylcholine, via phospholipase D. CONCLUSIONS: (A) Activation of PKC in the bovine iris sphincter leads to stimulation of adenylate cyclase and to an increase in cAMP formation. The cAMP formed inhibits IP3 production and muscle contraction. (B) PGF2 alpha desensitization results in adenylate cyclase activation, mediated through PKC. (C) PGF2 alpha desensitization could uncouple the receptor from the Gq and Gi proteins and enhance PG stimulation of adenylate cyclase activity through the Gs protein. (D) Uncoupling of the G proteins from the PG receptor and activation of PKC, both of which result in enhanced cAMP formation, may underlie the mechanism of PGF2 alpha desensitization. (E) These observations demonstrate "cross talk" between the two second messenger systems and their physiologic consequences.

Muscarinic cholinergic induced subsensitivity in rabbit iris-ciliary body. Effects on myo-inositol triphosphate accumulation, arachidonate release, prostaglandin synthesis, and contraction.

The relationship between desensitization of iris muscarinic acetylcholine receptors and the receptor-mediated phosphoinositide hydrolysis system was examined in rabbit eyes. Rabbits were treated topically in one eye, the other eye served as control, with 2% pilocarpine in three successive doses (2 X 50 microL) at 32, 24 and 6 hr prior to sacrifice. The polyphosphoinositide (PPI) response was measured in vitro in the iris both as loss of 32P-radioactivity from phosphatidylinositol 4,5-bisphosphate (PIP2) and as accumulation of myo-[3H]-inositol trisphosphate (IP3); arachidonic acid (AA) liberation was measured by radiochromatography; prostaglandin (PG) E2 release was measured by radiochromatography and radioimmunoassay; and myosin light chain (MLC) phosphorylation was measured with SDS-polyacrylamide gel electrophoresis. Desensitized tissues showed significant decreases in carbachol-induced PIP2 hydrolysis, IP3 accumulation, AA liberation, PGE2 synthesis and muscle contraction. These effects are specific to cholinergic receptors in the iris, since substance P-induced IP3 accumulation and PGE2 release, was not affected by the cholinergic desensitization. The cholinergic desensitization of the PPI response in the iris is reversible. These findings are in accord with our previous studies on alpha 1-adrenergic desensitization of the PPI response in this tissue, and they add further support to the hypothesis that changes in the activities of the receptor-mediated phosphoinositide hydrolysis system and its derived second messengers may underlie the mechanism of alpha 1-adrenergic and muscarinic cholinergic subsensitivity in the iris-ciliary body.