Effect of magnetized water on blood indices and histomorphometric parameters of intestinal sections of Japanese quail.

A two-way experimental design was used to demonstrate the physiological effects of magnetized water and sex on blood indices and histomorphometric parameters of Japanese quail intestine sections. Red blood cell count (RBCs), packed cell volume (PCV), hemoglobin concentration (Hb), thrombocytes, white blood cell count (WBCs), and WBC differentiation were investigated. A total of 450 unsexed Japanese quail were randomized into three groups (45/replicate; 3 replicates; 135/group). As a monitoring group, the first group was given untreated tap water to drink. The two others were consumed magnetized water that were subjected to an electrical magnetic field with a power of 1 Tesla (10,000 Gauss) and 2 Tesla (20,000 Gauss), respectively. The treatments had a significant (p ≤ 0.05) effect on thrombocytes and Hb. Sex showed significant (p ≤ 0.05) differences for RBCs and PCV at 42 days of age. At different ages, significant effects were observed on histomorphometric parameters of the Japanese quail intestinal tract. It may be inferred that the influence of magnetized water, up to 1 Tesla, was positive on the haematological and histomorphometric parameters of the Japanese quail intestinal tract by augmenting the haematological measurements, which were within a normal range and increasing the surface area of the villus.

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.

Induction of 8-hydroxydeoxyguanosine and ultrastructure alterations by silver nanoparticles attributing to placental transfer in pregnant rats and fetuses.

A quantitative assessment of the genotoxicity of silver nanoparticles (AgNPs) ascribed to its transplacental transfer and tissue distribution in pregnant rats was carried out in this study. A single intravenous (i.v.) injection of AgNPs with a size range from 4.0 to 17.0 nm was administered to pregnant rats at a dose of 2 mg/kg b.w. on the 19th day of gestation. Five groups beside control, each of the five rats were euthanized after 10 min, 1, 6, 12, or 24 h, respectively. The accumulation of nanoparticles (NPs) in mother and fetal tissues was quantified by inductively coupled plasma optical emission spectroscopy, where the highest accumulation level was recorded in maternal blood (0.523 µg/ml) after 24 h of administration. AgNPs induced accumulation in spleen tissue higher than placenta and fetal tissue homogenates. The data showed significantly detected levels of 8-hydroxydeoxyguanosine in all collected samples from administered animals compared with untreated individuals. Level of 8-OHdG in amniotic fluid exhibited the greatest values followed by maternal spleen, kidneys, and liver, respectively. Investigation by transmission electron microscope showed that the transfer of AgNPs through placental wall caused indentation of nuclei, clumped chromatin, pyknotic nuclei, and focal necrotic areas, while AgNPs appeared mainly accumulated in the macrophages of the spleen. Therefore, the data assume that the genotoxicity studies of AgNPs must be recommended during a comprehensive assessment of the safety of novel types of NPs and nanomaterials. Additionally, exposure to AgNPs must be prevented or minimized during pregnancy or prenatal periods.

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.

Evidence that a lipolytic enzyme--hematopoietic-specific phospholipase C-ß2--promotes mobilization of hematopoietic stem cells by decreasing their lipid raft-mediated bone marrow retention and increasing the promobilizing effects of granulocytes.

Hematopoietic stem/progenitor cells (HSPCs) reside in the bone marrow (BM) microenvironment and are retained there by the interaction of membrane lipid raft-associated receptors, such as the α-chemokine receptor CXCR4 and the α4ß1-integrin (VLA-4, very late antigen 4 receptor) receptor, with their respective specific ligands, stromal-derived factor 1 and vascular cell adhesion molecule 1, expressed in BM stem cell niches. The integrity of the lipid rafts containing these receptors is maintained by the glycolipid glycosylphosphatidylinositol anchor (GPI-A). It has been reported that a cleavage fragment of the fifth component of the activated complement cascade, C5a, has an important role in mobilizing HSPCs into the peripheral blood (PB) by (i) inducing degranulation of BM-residing granulocytes and (ii) promoting their egress from the BM into the PB so that they permeabilize the endothelial barrier for subsequent egress of HSPCs. We report here that hematopoietic cell-specific phospholipase C-ß2 (PLC-ß2) has a crucial role in pharmacological mobilization of HSPCs. On the one hand, when released during degranulation of granulocytes, it digests GPI-A, thereby disrupting membrane lipid rafts and impairing retention of HSPCs in BM niches. On the other hand, it is an intracellular enzyme required for degranulation of granulocytes and their egress from BM. In support of this dual role, we demonstrate that PLC-ß2-knockout mice are poor mobilizers and provide, for the first time, evidence for the involvement of this lipolytic enzyme in the mobilization of HSPCs.

A novel perspective on stem cell homing and mobilization: review on bioactive lipids as potent chemoattractants and cationic peptides as underappreciated modulators of responsiveness to SDF-1 gradients.

Hematopoietic stem progenitor cells (HSPCs) respond robustly to α-chemokine stromal-derived factor-1 (SDF-1) gradients, and blockage of CXCR4, a seven-transmembrane-spanning G(αI)-protein-coupled SDF-1 receptor, mobilizes HSPCs into peripheral blood. Although the SDF-1-CXCR4 axis has an unquestionably important role in the retention of HSPCs in bone marrow (BM), new evidence shows that, in addition to SDF-1, the migration of HSPCs is directed by gradients of the bioactive lipids sphingosine-1 phosphate and ceramide-1 phosphate. Furthermore, the SDF-1 gradient may be positively primed/modulated by cationic peptides (C3a anaphylatoxin and cathelicidin) and, as previously demonstrated, HSPCs respond robustly even to very low SDF-1 gradients in the presence of priming factors. In this review, we discuss the role of bioactive lipids in stem cell trafficking and the consequences of HSPC priming by cationic peptides. Together, these phenomena support a picture in which the SDF-1-CXCR4 axis modulates homing, BM retention and mobilization of HSPCs in a more complex way than previously envisioned.

Conditioning for hematopoietic transplantation activates the complement cascade and induces a proteolytic environment in bone marrow: a novel role for bioactive lipids and soluble C5b-C9 as homing factors.

We have observed that conditioning for hematopoietic transplantation by lethal irradiation induces a proteolytic microenvironment in the bone marrow (BM) that activates the complement cascade (CC). As a result, BM is enriched for proteolytic enzymes and the soluble form of the terminal product of CC activation, the membrane attack complex C5b-C9 (MAC). At the same time, proteolytic enzymes induced in irradiated BM impair the chemotactic activity of α-chemokine stromal-derived factor-1 (SDF-1). As SDF-1 is considered a crucial BM chemoattractant for transplanted hematopoietic stem/progenitor cells (HSPCs), we sought to determine whether other factors that are resistant to proteolytic enzymes have a role in this process, focusing on proteolysis-resistant bioactive lipids. We found that the concentrations of sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P) increase in the BM after conditioning for transplantation and that both S1P and, as we show here for the first time, C1P are potent chemoattractants for HSPCs. Next, we observed that C5-deficient mice that do not generate MAC show impaired engraftment of HSPCs. In support of a role for MAC in homing and engraftment, we found that soluble MAC enhances in a CR3 (CD11b/CD18)-dependent manner the adhesion of HSPCs to BM stromal cells and increases the secretion of SDF-1 by BM stroma. We conclude that an increase in BM levels of proteolytic enzyme-resistant S1P and C1P and activation of CC, which leads to the generation of MAC, has an important and previously underappreciated role in the homing of transplanted HSPCs.

Increased caspase-3 and altered expression of apoptosis-associated proteins, Bcl-2 and Bax in lichen planus.

BACKGROUND: Lichen planus (LP) is a chronic inflammatory disease of probable immune-based aetiology. The pathogenesis of LP is unclear, but apoptotic changes in epidermal (epithelial) cells have been reported. OBJECTIVE: To evaluate apoptosis in LP through studying caspase-3 expression and to determine whether the apoptosis-associated proteins Bcl-2 and Bax are significantly involved in the pathogenesis of LP. METHODS: In total, 25 lesional biopsy specimens [15 cutaneous LP (CLP) and 10 oral LP (OLP)] and 10 control specimens [5 normal skin and 5 normal oral mucosa] were studied using immunochemical methods for the expression of caspase-3, Bcl-2 and Bax proteins. RESULTS: Compared with controls, a significant increase in caspase-3 and Bax protein expressions were found in LP lesions. Basal cell expression of caspase-3 was positive in 14 cases (56%), and 12 cases (48%) showed mild expression. Caspase-3 expression in inflammatory infiltrate was positive in 13 cases (52%). Of these, 12 cases (48%) showed mild positivity. Bax was localized mostly to the upper prickle layer. Basal cell expression of Bcl-2 was negative in 18 (72%) cases, with no significant difference between patients with LP and controls. Bcl-2 was expressed in the inflammatory infiltrate in 15 cases of LP (60%), showing mild expression in 12 cases (48%). Compared with CLP, there was a significant increase in caspase-3 expression in OLP, despite the nonsignificant difference in Bcl-2 and Bax protein expressions by the epithelial cells. CONCLUSION: Increased caspase-3 and altered expression of Bcl-2 and Bax were found in LP, indicating the possible involvement of these proteins in the pathogenesis of the disease. The observed increase in apoptosis in OLP compared with CLP might explain the difference in clinical behaviour that distinguishes these LP variants.

Association of cytokine gene polymorphism with susceptibility and clinical types of leprosy.

BACKGROUND: Both pro- and anti-inflammatory cytokines are implicated in development and prognosis of leprosy so the genetic regulation of such cytokines could play an important role. OBJECTIVE: . This study was planned for testing the association of cytokine gene polymorphisms with susceptibility and clinical types of leprosy among Egyptian cases. SUBJECTS: This study included 47 cases (29 men, 18 females, mean age = 46.3 years) with leprosy in addition to 98 healthy unrelated controls (52 males, 46 females, mean age = 44.9 years). Cases were recruited from Leprosy Clinics, Delta region of Egypt. Cases were classified into paucibacillary (PB) (n = 17; 10 males, 7 females; mean age 42.6 years) and multibacillary (MB) (n = 29; 19 males, 10 females; mean age 43.9 years). METHODS: For all cases and controls, DNA was extracted and amplified using polymerase chain reaction with sequence specific primers (PCR-SSP) for detection of single nucleotide polymorphisms (SNPs) in the promoter regions of cytokine genes, TNF-α-308 (G/A), IL-10-1082(G/A), IL-6-174(G/C) as well as IL-1RaVNTR in intron 2 of the gene. RESULTS: COMPARED TO CONTROLS, ALL CASES HAVE SHOWN INCREASED FREQUENCY OF HOMOZYGOUS GENOTYPES : IL-10-1082 (GG) (Odds ratio 6.6, P <0.05), homozygous TNF-α-308 (GG) (Odds ratio =3.23), and homozygous IL-1Ra (11) (Odds ratio = 3.6, P<0.05) with increased frequency of IL10 G and ILRa 1 alleles (P<0.05). BP subgroup showed increased frequency of homozygous IL-10-1082 (GG) (Odds ratio = 18.6, P<0.05) with increased frequency of IL10 G allele (P<0.05). On the other hand, MB subgroup showed increased frequency of homozygous TNF-α-308 (GG) (Odds ratio = 5.84, P<0.05) and homozygous IL-1Ra (11) (Odds ratio = 4, P<0.05) with increased frequency of IL-1Ra 1 allele (P<0.05). There is predominance for heterozygous IL-6-174 (G/C) polymorphism in all studies patient subgroups as well as controls with no significant difference among them. CONCLUSION: Genetic polymorphisms related to TNF-α-308 and IL-10-1082 and IL-1Ra may be used as genetic markers for susceptibility and clinical outcome of leprosy among Egyptian cases from the Nile Delta.

Biocontrol potential of salinity tolerant mutants of Trichoderma harzianum against Fusarium oxysporum

A exposição de uma cepa selvagem de Trichoderma harzianum à irradiação gama induziu dois mutantes tolerantes a sal (Th50M6 e Th50M11). Em condições salinas, os dois mutantes foram muito superiores à cepa selvagem em relação à velocidade de multiplicação, esporulação e eficiência contra Fusarium oxysporum, o agente causador da doença wilt do tomate. Os mutantes tolerantes foram capazes de multiplicação e esporulação em meio de cultura contendo NaCl até 69 mM. Em comparação à cepa selvagem, os dois mutantes possuíam conteúdo mais elevado de prolina e hidroxiprolina, conteúdo de sódio superior ao de potássio, cálcio ou magnésio e conteúdo elevado de fenóis totais. A análise eletroforética das proteínas totais solúveis no mutante Th50M6 apresentou bandas diferentes acumuladas em resposta a NaCl 69 mM. Os resultados também indicaram que os mutantes produzem alguns metabólitos ativos como quitinases, celulases, b-galactosidades e antibióticos como tricodermina, gliotoxina e gliovirina. Os mutantes de Trichoderma reduziram significativamente a incidência da doença e melhoraram o rendimento e o conteúdo de minerais do tomate tanto em condições salinas como não-salinas e também em condições naturais e de infestação. Quando comparados à cepa selvagem, os mutantes de Trichoderma foram também mais eficientes em diminuir o crescimento de F. oxysporum na rizosfera.A densidade populacional de ambos mutantes na rizosfera excedeu muito a da cepa selvagem.

Cardiac stem cell therapy for myocardial regeneration. A clinical perspective.

Myocardial infarction and other pathologic conditions of the heart result in loss of cardiomyocytes, scar formation, ventricular remodeling, and eventually heart failure. Since pharmacologic and interventional strategies fail to regenerate dead myocardium, heart failure continues to be a major health problem worldwide. Recent studies in animal models of myocardial infarction and heart failure have demonstrated that various subsets of adult primitive cells can regenerate functional cardiomyocytes and cardiac vasculature with improvement in cardiac structure and function. Small clinical trials of cell therapy in patients with myocardial infarction and ischemic cardiomyopathy have recapitulated these beneficial effects in humans with infarct size reduction and improvement in ejection fraction, myocardial perfusion, and wall motion. Several phenotypically distinct cell populations have been utilized for cardiac regeneration, and the relative merits of one cell over another remain to be determined. The recent discovery of adult cardiac stem cells has sparked intense hope for myocardial regeneration with cells that are from the heart itself and are thereby inherently programmed to reconstitute cardiac tissue. The purpose of this review is to summarize the evidence regarding the feasibility of cardiac repair in humans via adult stem/progenitor cells, and to discuss the potential utility of cardiac stem cells for therapeutic myocardial regeneration.

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.

Pattern of primary nocturnal enuresis in primary school children (first grade) in assiut city

Objectives :To assess the prevalence of nocturnal enuresis in primary school children; first grade (6-7 years old) in Assiut City and study its pattern and risk factors. Patients and Methods: A random cross-sectional study including 1519 children was conducted in 10 primary schools in Assiut City throughout a period of six months. A self-administered questionnaire was completed voluntarily by the parents. Children reporting two or more incidences of nocturnal enuresis per month during the past year were considered positive repliers and were subjected to further evaluation. The control group consisting of 100 age-matched non-enuretic children presenting for other urologic or non-urologic problems mandating a plain abdominal X-ray was subjected to the same evaluation. In these children the possible detection of spina bifida was the point of concern. Results: The response rate to the questionnaire was 79. The prevalence of enuresis was 20.2. Out of the enuretic children; 87.6complained of nocturnal enuresis only; 72.3of them were primary enuretics. Monosymptomatic enuresis was observed in 46.3of the cases. Marked enuresis (every night) affected 53.7of the total number of enuretic children. Statistically significant risk factors were deep sleep and a high educational level of the parents. Primary nocturnal enuresis was insignificantly associated with a positive family history; family size or birth rank. In primary nocturnal enuretics the results of the urogenital and neurological examinations were normal in 90.5and 80.6; respectively; and only 4received a specific treatment. A large amount of post-void residual urine was observed in 7.4. Spina bifida occulta was observed in 10.8of primary nocturnal enuretics and in 11 of the control group. Conclusions: This is the first large population-based study of the prevalence of age-related enuresis in Assiut; but it does not differ much from those reported in other parts of the world. Enuretic children and their parents are mildly concerned about enuresis. Investigations for monosymptomatic primary nocturnal enuresis are not of considerable diagnostic value or cost effectiveness

Activation of p42/p44 mitogen-activated protein kinase and contraction by prostaglandin F2alpha, ionomycin, and thapsigargin in cat iris sphincter smooth muscle: inhibition by PD98059, KN-93, and isoproterenol.

In the present study we investigated the cross talk between the Ca2+ mobilization pathway and the mitogen-activated protein (MAP) kinase pathway and contraction in the cat iris sphincter smooth muscle. Three Ca2+-mobilizing agonists, namely, prostaglandin F2alpha (PGF2alpha), ionomycin, and thapsigargin, and three specific inhibitors, PD98059, a p42/p44 MAP kinase inhibitor; KN-93, a Ca2+-calmodulin-dependent protein kinase II (CaMKII) blocker; and isoproterenol, a cAMP-elevating agent, were used. Changes in tension in response to the agonists were recorded isometrically and MAP kinase phosphorylation and activation were monitored by Western blotting and by in situ myelin basic protein phosphorylation, respectively. We found that 1) stimulation of the sphincter muscle with PGF2alpha, ionomycin, or thapsigargin resulted in rapid phosphorylation and activation of p42/p44 MAP kinase and contraction; and 2) treatment of the muscles with PD98059, KN-93, or isoproterenol resulted in inhibition of the Ca2+-mobilizing agonist-induced responses. The contractile responses induced by PGF2alpha, ionomycin, and thapsigargin were (mg of tension/mg of wet weight tissue) 15.2, 15.4, and 16.2, respectively; the increases in MAP kinase phosphorylation by these agonists were 228, 203, and 190%, respectively; and the increases in MAP kinase activation by the agonists were 212, 191, and 162%, respectively. The stimulatory effects of the agonists on contraction and on MAP kinase phosphorylation and activation were blocked by preincubation of the muscle with PD98059, KN-93, or isoproterenol. These data demonstrate that in the iris sphincter phosphorylation and activation of p42/p44 MAP kinases by PGF2alpha, ionomycin, or thapsigargin require intracellular Ca2+ either from extracellular sources or from internal stores, that CaMKII plays an important role in the regulation of contraction, that CaMKII acts upstream of MAP kinase to control its activation, and that the MAP kinase signaling pathway can play a significant role in mediating the cellular effects of these Ca2+-mobilizing agonists.

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-beta1-4 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.

Effects of prostaglandin F(2alpha)and carbachol on MAP kinases, cytosolic phospholipase A(2)and arachidonic acid release in cat iris sphincter smooth muscle cells.

The signal transduction pathways initiated by Ca(2+)-mobilizing agonists, such as prostaglandin F(2alpha)(PGF(2alpha)) and carbachol (CCh), leading to activation of cytosolic phospholipase A(2)(cPLA(2)) and arachidonic acid (AA) release in a wide variety of tissues remain obscure. To further define the role of protein kinases in receptor mediated stimulation of cPLA(2)and consequently AA release we have investigated the role of mitogen-activated protein (MAP) kinases and protein kinase C (PKC) in PGF(2alpha)- and CCh-induced cPLA(2)phosphorylation and AA release in cat iris sphincter smooth muscle (CISM) cells. The cells were prelabeled with [(3)H]AA for 24 hr and incubated in the absence or presence of the agonist for 5-10 min as indicated. MAP kinases activities and cPLA(2)phosphorylation were determined in immunoprecipitates obtained by using anti-p38 MAP kinase and anti-cPLA(2)antibodies. We found that: (a) PGF(2alpha)and CCh increased p38 MAP kinase activity by 197 and 215%, respectively, and increased p42/p44 MAP kinase activity by 200 and 125%, respectively. (b) SB202190, a p38 MAP kinase specific inhibitor, inhibited PGF(2alpha)- and CCh-induced cPLA(2)phosphorylation by 92 and 85%, respectively, and AA release by 62 and 78%, respectively. (c) PD98059, a p42/p44 MAP kinase inhibitor, inhibited CCh-induced cPLA(2)phosphorylation by 70% and AA release by 71%, but had no effect on that of PGF(2alpha). (d) Inhibition of PKC activity by RO 31-8220 inhibited both PGF(2alpha)- and CCh-stimulation of p38 MAP kinase, p42/p44 MAP kinases and cPLA(2)phosphorylation. We conclude from these results that in CISM cells PGF(2alpha)-induced cPLA(2)phosphorylation and AA release is mediated through p38 MAP kinase, but not through p42/p44 MAP kinases, whereas that of CCh is mediated through both p38 MAP kinase and p42/p44 MAP kinases. These effects of PGF(2alpha)and CCh are regulated by the MAP kinases in a PKC-dependent manner. Studies aimed at elucidating the role of protein kinases in the coupling mechanism between the activation of PGF(2alpha)and muscarinic receptors, and the stimulation of cPLA(2)and AA release in the smooth muscles of the iris-ciliary body will provide important information about the role of protein kinases signaling pathways in smooth muscle function, as well as about the mechanism of the intraocular pressure-lowering effects of PGF(2alpha)and its analog, latanoprost, in glaucoma therapy.

Role of protein kinase C alpha and mitogen-activated protein kinases in endothelin-1-stimulation of cytosolic phospholipase A2 in iris sphincter smooth muscle.

We have investigated the roles of protein kinase C (PKC) and mitogen-activated protein kinases (MAPK) in the phosphorylation and activation of cytosolic phospholipase A2 (cPLA2) in endothelin-1- (ET-1) stimulated cat iris sphincter smooth muscle (CISM) cells. We found that in these cells both PKC and p38 MAP kinases play a critical role in ET-1-induced cPLA, phosphorylation and arachidonic acid (AA) release. Our findings indicate that stimulation of the endothelin-A- (ET(A)) receptor leads to: (1) activation of Gq protein which stimulates phospholipase C to hydrolyze the polyphosphoinositide PIP, into diacylglycerol (DAG) and inositol trisphosphate (IP3), the DAG may then activate PKC to phosphorylate and activate cPLA2; and (2) activation of Gi protein, which, through a series of kinases, leads to the stimulation of p38 MAPK and subsequently to phosphorylation and activation of cPLA2. The ability of the activated ET(A)-receptor, which is coupled to both Gq and Gi proteins, to recruit and activate this complex signal transduction mechanism remains to be clarified.

Mitogen-activated protein kinase inhibitors suppress prostaglandin F(2alpha)-induced myosin-light chain phosphorylation and contraction in iris sphincter smooth muscle.

The purpose of this study was to investigate the potential role of mitogen-activated protein (MAP) kinase in contraction by monitoring MAP kinase phosphorylation (activation) and contraction during agonist stimulation of cat iris sphincter smooth muscle. Changes in tension in response to prostaglandin F(2alpha), latanoprost, a prostaglandin F(2alpha) analog used as an anti-glaucoma drug, and carbachol were recorded isometrically, and MAP kinase activation was monitored by Western blot using a phosphospecific p42/p44 MAP kinase antibody. We found that treatment of the muscle with 2'-Amino-3'-methoxyflavone (PD98059) (10 microM), a specific inhibitor of MAP kinase kinase (MEK), inhibited significantly prostaglandin F(2alpha)- and latanoprost-induced phosphorylation and contraction, but had little effect on those evoked by carbachol. Prostaglandin F(2alpha) increased MAP kinase phosphorylation in a concentration-dependent manner with EC(50) value of 1.1 x 10(-8) M and increased contraction with EC(50) of 0.92 x 10(-9) M. The MAP kinase inhibitors PD98059, Apigenin and 1,4-Diamino-2,3-dicyano-1, 4bis(2-aminophenylthio)butadiene (UO126) inhibited prostaglandin F(2alpha)-induced contraction in a concentration-dependent manner with IC(50) values of 2.4, 3.0 and 4.8 microM, respectively. PD98059 had no effect on prostaglandin F(2alpha)- or on carbachol-stimulated inositol-1,4,5-trisphosphate (IP(3)) production. In contrast, the MAP kinase inhibitor inhibited prostaglandin F(2alpha)-induced myosin-light chain (MLC) phosphorylation, but had no effect on that of carbachol. N-[2-(N-(4-Chloro-cinnamyl)-N-methylaminomethyl)phenyl]-N-[2- hydroxyethyl]-4-methoxybenzenesulfonamide (KN-93) (10 microM), a Ca(2+)-calmodulin-dependent protein kinase inhibitor, and Wortmannin (10 microM), an MLC kinase inhibitor, inhibited significantly (by 80%) prostaglandin F(2alpha)- and carbachol-induced contraction. It can be concluded that in this smooth muscle p42/p44 MAP kinases are involved in the mechanism of prostaglandin F(2alpha)-, but not in that of carbachol, induced contraction. In addition, these data clearly indicate that the stimulation of the iris sphincter with prostaglandin F(2alpha) and carbachol activate two distinct pathways, the MAP kinase pathway and the Ca(2+) mobilization pathway.

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.