Effect of low dose metronomic therapy on MCF-7 tumor cells growth and angiogenesis. Role of muscarinic acetylcholine receptors.

The non-neuronal cholinergic system refers to the presence of acetylcholine, choline acetyltransferase, acetylcholinesterase and cholinergic receptors, nicotinic and muscarinic (mAChRs) expressed in non-neuronal cells. The presence of mAChRs has been detected in different type of tumor cells and they are linked with tumorigenesis. We had previously documented the expression of mAChRs in murine and human mammary adenocarcinomas and the absence of these receptors in normal mammary cells of the same origins. We also demonstrated that mAChRs are involved in breast cancer progression, pointing to a main role for mAChRs as oncogenic proteins. Since the long term treatment of breast cancer cells with the muscarinic agonist carbachol promoted cell death, here we investigated the ability of low doses of this agonist combined with paclitaxel (PX), a taxane usually administered to treat breast cancer, to inhibit the progression of human MCF-7 tumor cells. We demonstrated that PX plus carbachol reduced cell viability and tumor growth in vitro probably due to a down-regulation in cancer stem cells population and in the expression of ATP "binding cassette" G2 drug extrusion pump; also a reduction in malignant-induced angiogenesis was produced by the in vivo administration of the mentioned combination in a metronomic schedule to MCF-7 tumor-bearing NUDE mice. Our results confirm that mAChRs could be considered as therapeutic targets for metronomic therapy in breast cancer as well as the usefulness of a muscarinic agonist as repositioning drug in the treatment of this type of tumors.

Treatment with LPS plus INF-γ induces the expression and function of muscarinic acetylcholine receptors, modulating NIH3T3 cell proliferation: participation of NOS and COX.

BACKGROUND AND PURPOSE: LPS and IFN-γ are potent stimuli of inflammation, a process in which fibroblasts are frequently involved. We analysed the effect of treatment with LPS plus IFN-γ on the expression and function of muscarinic acetylcholine receptors in NIH3T3 fibroblasts with regards to proliferation of these cells. We also investigated the participation of NOS and COX, and the role of NF-κB in this process. EXPERIMENTAL APPROACH: NIH3T3 cells were treated with LPS (10 ng·mL(-1)) plus IFN-γ (0.5 ng·mL(-1)) for 72 h (iNIH3T3 cells). Cell proliferation was evaluated with MTT and protein expression by Western blot analysis. NOS and COX activities were measured by the Griess method and radioimmunoassay respectively. KEY RESULTS: The cholinoceptor agonist carbachol was more effective at stimulating proliferation in iNIH3T3 than in NIH3T3 cells, probably due to the de novo induction of M3 and M5 muscarinic receptors independently of NF-κB activation. iNIH3T3 cells produced higher amounts of NO and PGE2 than NIH3T3 cells, concomitantly with an up-regulation of NOS1 and COX-2, and with the de novo induction of NOS2/3 in inflamed cells. We also found a positive feedback between NOS and COX that could potentiate inflammation. CONCLUSIONS AND IMPLICATIONS: Inflammation induced the expression of muscarinic receptors and, therefore,stimulated carbachol-induced proliferation of fibroblasts. Inflammation also up-regulated the expression of NOS and COX-2, thus potentiating the effect of carbachol on NO and PGE2 production. A positive crosstalk between NOS and COX triggered by carbachol in inflamed cells points to muscarinic receptors as potential therapeutic targets in inflammation.

Nitric oxide synthase-cyclooxygenase interactions are involved in tumor cell angiogenesis and migration.

Nitric oxide (NO), produced by distinct nitric oxide synthase (NOS) isoforms, and prostaglandins generated by expression of cyclooxygenases are important mediators in tumor progression. Previous studies have shown that NO can influence the formation of prostaglandin E2 (PGE2). We provide evidence that NO, derived from iNOS and eNOS activity in LMM3 murine mammary adenocarcinoma cell line, is involved in tumor angiogenesis and in tumor cell migration. LMM3 cells that also stimulate their neovascularization activity and migration liberate high basal amounts of PGE2. There is large amount of evidence that postulates positive regulatory interactions between NOS and cyclooxygenase (COX) isoforms. We here show that, in the LMM3 cell line, while PGE2 exerts a positive modulation on NOS activity, NO closes the loop with a negative feed back on COX activity. We also provide evidence of a positive regulatory effect of protein tyrosine kinases on NOS as well as on COX enzymatic functions affecting tumor induced angiogenesis and cell migration.

Parasympathetic modulation of amylase secretion by IFN gamma in murine submandibular glands.

IFN gamma is a pleiotropic cytokine that exerts immunologic and non-immunologic functions. We show here that at low doses (10 U/ml), it stimulates amylase secretion in murine submandibular glands (SMG) "via" muscarinic receptor activation, comparable to that produced by the muscarinic agonist carbachol. Both effects are blocked by atropine. NG-monomethyl-L-arginine (L-NMMA) and EGTA inhibited the cytokine effect on amylase secretion, involving the participation of a calcium-dependent isoform of nitric oxide synthase (NOS). We confirm NOS activation because IFN gamma stimulates nitrite production and enzyme activity in SMG. Carbachol (10(-7) M) did not modify basal nitric oxide production. In addition, both IFN gamma and carbachol increase prostaglandin E2 production in SMG, but while indomethacin potentiates IFN gamma effect on amylase secretion, it blunted amylase secretion exerted by carbachol. Thus, IFN gamma and carbachol stimulate IFN gamma secretion on SMG in a dose-dependent manner. Our results are pointing to neuroregulatory functions of IFN gamma in murine SMG, because it regulates its own levels in oral cavity, perhaps to exert a local immuno-surveillance.

Participation of nitric oxide synthase and cyclo-oxygenase in the signal transduction pathway of ileal muscarinic acetylcholine receptors.

Parasympathetic activation of ileal motility is essential for intestinal physiology. We have previously demonstrated that carbachol activates muscarinic acetylcholine receptors (mAChR) of rat intestine and stimulates ileal motility via phospholipase C. This activation induces phosphoinositide turnover and intracellular calcium mobilization. We show here that carbachol stimulation of rat ileal motility is potentiated by the nitric oxide synthase (NOS) inhibitor N(G)-monomethyl arginine. Thus, we confirm that carbachol increases, in a dose-dependent manner, the activity of a NOS isoform that depends on calcium-calmodulin binding. Its product, nitric oxide (NO), activates not only guanylyl cyclase, inducing cGMP synthesis, but also cyclo-oxygenase, producing prostaglandin E(2). The prostanoid probably cooperates with NO to induce ileal smooth muscle relaxation.

Differential activation of nitric oxide synthase through muscarinic acetylcholine receptors in rat salivary glands.

Muscarinic receptors play an important role in secretory and vasodilator responses in rat salivary glands. Nitric oxide synthase (NOS) appears to be one of the multiple effectors coupled to muscarinic receptors in both submandibular and sublingual glands although some differences have been found depending on the gland studied. First, submandibular glands had a lower basal activity of nitric oxide synthase than sublingual glands and the concentration-response curve for carbachol was bell-shaped in the former but not in sublingual glands. Second, cGMP levels displayed a similar profile to that observed for NOS activity in both glands. Third, protein kinase C also coupled to muscarinic receptor activation in the glands might have a regulatory effect on nitric oxide production since its activity was higher in basal conditions in submandibular than sublingual glands and it also increased in the presence of the agonist at a concentration that inhibited NOS activity in submandibular glands. The effects appear to be partly related to the expression of a minor population of M(1) receptors in submandibular glands absent in sublingual as determined in binding and signaling experiments with the muscarinic receptor antagonist pirenzepine.

Tyrosine kinase regulatory action on ileal muscarinic effects of IFN-gamma.

Interferon-gamma (IFN-gamma) is a pleiotropic cytokine that has a large number of immunologic and nonimmunologic functions. We have described that IFN-gamma could activate muscarinic cholinergic receptors (mAchR) of rat intestine, stimulating ileal motility. We also observed that mAchR activation induced inhibition of cAMP levels and stimulation of cGMP formation. The objectives of our work were to clarify the signal transduction pathways involved in regulation of ileal motility through mAchR activation by IFN-gamma. Our results demonstrate that this cytokine produces an ileal cholinergic response through tyrosine kinase activity. The activation of tyrosine kinase mediates ileal contractility, phosphoinositide hydrolysis by phospholipase C, nitric oxide synthase via protein kinase C, and cGMP synthesis. The increment in ileal motility is probably due to hyperproduction of prostaglandin E2 (PGE2) by ileal tissue. This prostanoid is an important mediator because it stimulates ileal motility. We conclude that IFN-gamma not only immunomodulates the gut microenvironment but also exerts a local nonimmunologic regulation on intestinal motility.

Protein kinase C regulates NO-cGMP pathway in muscarinic receptor activation by HIV+-IgA.

In this work we demostrate that IgA purified from HIV infected patients recognizes a band with a molecular weight corresponding to radiolabelled ileal muscarinic acethylcholine receptors (mAChR) by immunoblotting. HIV+-IgA triggers the signals that are the consequence of mAChR stimulation in the intestine, regulating protein kinase C (PKC) and nitric oxide synthase (NOS) activity as well as 3',5'-cyclic guanosine monophosphate (cGMP) formation. On the one hand PKC activation by HIV+-IgA induces NOS inhibition and as a consequence, low amounts of NO that could improve local immunosuppression in the intestine; on the other hand HIV+-IgA stimulates cGMP production which could potentiate ileal motility and loss of water/electrolytes involved in intestinal damage in AIDS.

Role of arachidonic acid metabolites in the action of a beta adrenergic agonist on human monocyte phagocytosis.

The mechanisms by which beta adrenergic stimulation regulates phagocytosis of Candida albicans by human peripheral monocytes (HPM) are characterized. Isoproterenol (ISO) inhibits phagocytosis in a concentration-dependent manner. This effect was blunted by propranolol, inhibitors of phospholipase A2 (PLA2), cyclooxygenase and verapamil, pointing to a participation of arachidonic acid (AA) metabolites and calcium in the phenomenon. Prostaglandin E2 (PGE2) and dibutyryl cyclic AMP (db-cAMP) also exerted the same inhibitory effect on phagocytosis. ISO interacts with beta adrenergic receptors of HPM increasing PGE2 and cAMP. We conclude that the mechanisms by which beta adrenergic stimulation regulates phagocytosis of Candida albicans by HPM appear to be secondary to beta adrenoceptor-mediated hydrolysis of AA accompanied by an increase in PGE2 generation and cAMP production. Both PGE2 and cAMP could act as mediators of the inhibitory action of beta agonists on the HPM-phagocytosis process.

IgA from HIV+ haemophilic patients triggers intracellular signals coupled to the cholinergic system of the intestine.

IgA was obtained from HIV-infected haemophilic patients and the intracellular signals triggered by its reaction with isolated rat intestinal strips were studied. HIV+ IgA stained intestinal microvilli with a granular immunofluorescence pattern and bound to the muscarinic acetylcholine receptor (mAChR), displacing the specific muscarinic cholinergic antagonist QNB in a non-competitive manner. It triggered the signals that are the consequence of mAChR stimulation in the intestine. Thus, it decreased cAMP synthesis and increased guanosine 3':5'-cyclic monophosphate (cGMP) formation and phosphoinositide (PI) turnover of the intestine. In addition, it stimulated prostaglandin E2(PGE2) synthesis by intestinal strips. Through its effect on PGE2 synthesis, HIV+ IgA could have a dual action. On the one hand, it could enhance immunosuppression at a local level, favouring pathogen growth and subsequent intestinal dysfunction. On the other hand, PGE2 could directly increase intestinal motility and electrolyte/fluid loss. Both effects could be involved in intestinal damage in AIDS.

Intracellular signals coupled to different rat ileal muscarinic receptor subtypes.

Taking into account that the activation of different subtypes of ileal muscarinic acetylcholine receptors (mAChR) regulate gut functions such as tone, motility, and electrolyte secretion, we characterized the expression of mAChR in ileal-purified membranes. We also studied intracellular signals triggered by mAChR activation. Binding parameters obtained from saturation assays with the nonselective tritiated muscarinic antagonist, quinuclidynil benzilate ([3H]-QNB), were maximal number of binding sites (Bmax): 30 +/- 2 fmol/mg prot and dissociation constant (Kd): 0.2 +/- 0.03 nM. The competitive inhibition of [3H]-QNB specific binding by various nonlabelled muscarinic antagonists was measured and the rank order of potency was: atropine (ATROP) > 4-DAMP > AF-DX 116 > pirenzepine (PZ). The activation of mAChR by carbachol (CARB) increased ileal motility in a concentration-dependent manner (EC50 2 x 10[-7] M). The antagonists' order of potency to displace dose-response curve of CARB was: ATROP > 4-DAMP > AF-DX116 > PZ. Optimal concentration of CARB on ileal strips increased phosphoinositide turnover and cGMP levels by activating ml receptor subtype and decreased isoproterenol (ISO) stimulated levels of cAMP due to M2 receptor activation. We can conclude that the activation of different mAchR subtypes triggers different intracellular signals that could regulate intestinal tone and motility.

Induction of ileum muscarinic cholinoceptor signal transduction pathways by rat interferon-gamma.

We have recently shown that rat interferon-gamma (IFN gamma) can trigger a cholinergic muscarinic response upon interaction with isolated rat atria, decreasing both the contractile tension and cAMP synthesis, and increasing cGMP production. Because the intestine is rich in cholinergic muscarinic receptors, and IFN gamma-producing cells are normal components of the mucosal immune system, we investigated whether IFN gamma could also modulate the muscarinic cholinergic activity of the intestine. Our results indicate that IFN gamma may interact with ileum cholinergic muscarinic receptors by inhibiting binding of the radiolabelled antagonist quinuclidinyl benzylate (3H-QNB), decreasing cAMP formation and increasing cGMP synthesis. These effects are associated with increased mechanical activity of the isolated ileum strips, and can be blunted by pretreatment of the tissue with atropine.

Role of prostaglandin E2 in alterations of the beta-adrenergic system from rat eclamptic uterus.

The inotropic effect of isoproterenol, as well as the beta-adrenoceptor population, was measured in pregnant uterine tissue from female spontaneous hypertensive rats (SHR) (control group: C) and female SHR that were grafted with skin from Holtzman male rats (eclamptic group: E). The Kd value of the concentration-response curve of isoproterenol was higher for uteri from E rats than C rats. This phenomenon was not accompanied by a modification in the expression of beta-adrenoceptors. Inhibition of the synthesis of prostaglandins prevented the hyporeactivity to isoproterenol during eclampsia. Moreover, uteri from E rats generated and released greater amounts of prostaglandin E2 (PGE2) than uteri from C rats, even in the presence or absence of isoproterenol. In addition, whereas isoproterenol administered alone increased basal cyclic AMP (cAMP) production from C uteri, PGE2 administered alone enhanced cAMP production in E uterine tissue. These results suggest that the decrease in beta-adrenergic response to the agonist in E rats is ascribed to PGE2 production. The abnormal reactivity to the beta-agonist could be associated with a heterologous desensitization of uterine beta-adrenoceptors exerted by PGE2 overload in uteri from E rats. These results bear directly on the regulation of uterine motility during pregnancy, since an impaired response to beta-adrenergic innervation could lead to increased uterine motility, impairing the maintenance of pregnancy.

Uterine beta-adrenergic receptors in allogeneic and syngeneic pregnancy.

Two different mechanisms of regulation in uterine beta-adrenergic receptors in allogeneic pregnancy (AP) and syngeneic pregnancy (SP) are described in this work. Firstly we noted changes in beta-adrenergic sensitivity to isoproterenol in AP, while in SP differences in uterine reactivity to isoproterenol were found. In binding assays with the beta-antagonist [3H]dihydroalprenolol, changes in beta-receptor affinity were seen in AP, while in SP the maximal number of binding sites is altered. In addition, there are differences in uterine concentrations of cyclic AMP in both types of pregnancies as well as in the nucleotide response to isoproterenol. The differences in the reactivity and expression of uterine beta-adrenoceptors in both types of pregnancies may be due to a distinctive immunological role of the semiallogeneic fetus in AP.

Allopregnant IgG binds and modulates the activity of uterine beta adrenoceptors.

Female Balb/c mice were mated to C3H male mice, to obtain an allopregnant IgG. This allopregnant IgG con activate beta-adrenoceptors of C3H uterus. Here we show that allopregnant IgG interacts with beta-adrenoceptors in uterus of C3H mice and produces a decrease of the spontaneous motility and an increase of the intracellular cAMP concentration. Both effects are blocked by propranolol, a beta-adrenergic antagonist. Allopregnant IgG interferes with the binding of a specific beta adrenergic radioligand 3H-dihydroalprenolol behaving as a non competitive inhibitor. All these data indicate that allopregnant IgG could modulate the activity of uterine beta-adrenoceptors.

Allopregnant IgG binds and modulates the activity of uterine beta adrenoceptors

Ratones hembra Balb/c fueron apareados con machos C3H para obtener de las hembras una IgG de preñez alogeneica. Esta IgG activa los receptores ß adrenérgicos presentes en el útero de hembras C3H. En este trabajo se muestra que la IgG obtenida de hembras en alopreñez al interactuar con el receptor ß adrenérgico uterino de hembras C3H produce una inhibición de la motilidad espontánea uterina e incrementa los niveles intracelulares de AMPc. Ambos efectos son bloqueados por el antagonista ß adrenérgico propranolol. Por otra parte la IgG de preñez alogeneica interfiere la unión del radioligando ß específico 3H dihidroalprenolol comportándose como un inhibidor no competitivo. Los datos indican que esta IgG sería capaz de modular la actividad funcional de los receptores ß adrenérgicos uterinos

Differential PGE2 and cAMP production by allogeneic and syngeneic pregnant mice uteri.

Prostaglandin E2 (PGE2) and cAMP production in allogeneic and syngeneic pregnant mice uteri, were measured in relation to the ratio of plasma estrogen/progesterone levels. PGE2 generation by allopregnant uteri varied with the days of pregnancy. The increment of the prostanoid coincided with the increase in plasma estrogen concentration, whereas the decrement of its production was in parallel with the increment of plasma progesterone. The syngeneic pregnant uterus was unable to increase the release of PGE2 above basal values during the whole pregnancy. The rise of PGE2 production by the allogeneic pregnant uterus was correlated with an increase in cAMP levels. It is proposed that the pregnant mouse uterus increases its ability to release PGE2 in response to an ovarian steroids.

Functional alterations of murine peritoneal macrophages during pregnancy.

We have studied some functional characteristics of murine peritoneal macrophages (MOp) related to hormonal changes produced during pregnancy. Maximal expression of Ia antigen and release of interleukin 1 (IL1) were observed during the first week of pregnancy (implantation), when the highest peak of estradiol was produced. Both Ia antigen expression and IL1 levels progressively decreased as gestation advanced. Inversely, MOp capability to phagocyte and reduce nitro blue tetrazolium (NBT) was diminished at the beginning of pregnancy but returned to normal values in the last week. Sexual steroid levels (estradiol and progesterone) were inversely related, and the release of prostaglandin E2 (PGE2) by MOp decreased when progesterone levels increased. Although PGE2 production had no evident relation with Ia antigen expression and IL1 activity during the first and second weeks of pregnancy, the increment in PGE2 values and percentages of NBT+ cells could indicate a different stage of macrophage activation. These results suggest a possible hormonal regulation of macrophage functionality.

Sympathetic control of specific and non-specific immune response.

Following antigenic stimulation with specific (alloantigen) and non-specific (sheep red blood cells (SRBC) and lipopolysaccharide (LPS] antigens the expression of beta-adrenergic receptors and the interference of immune sera IgG, on uterine smooth muscle beta-adrenoceptor-specific ligands were studied. The binding of alloimmune IgG to beta-adrenoceptors appeared to be specific, because immunization with SRBC and LPS did not induce anti-beta-adrenoceptors antibodies. On the contrary, the decrease in beta-adrenergic receptor expression was observed even with alloantigens and with the conventional antigenic challenge, suggesting that this phenomenon could be a non-specific immunoregulatory mechanism.