The Piroplasmida Babesia, Cytauxzoon, and Theileria in farm and companion animals: species compilation, molecular phylogeny, and evolutionary insights.

The order Piroplasmida, including the genera Babesia, Cytauxzoon, and Theileria is often referred to as piroplasmids and comprises of dixenous hemoprotozoans transmitted by ticks to a mammalian or avian host. Although piroplasmid infections are usually asymptomatic in wild animals, in domestic animals, they cause serious or life-threatening consequences resulting in fatalities. Piroplasmids are particularly notorious for the enormous economic loss they cause worldwide in livestock production, the restrictions they pose on horse trade, and the negative health impact they have on dogs and cats. Furthermore, an increasing number of reported human babesiosis cases are of growing concern. Considerable international research and epidemiological studies are done to identify existing parasite species, reveal their phylogenetic relationships, and develop improved or new drugs and vaccines to mitigate their impact. In this review, we present a compilation of all piroplasmid species, isolates, and species complexes that infect domestic mammals and which have been well defined by molecular phylogenetic markers. Altogether, 57 taxonomic piroplasmid entities were compiled, comprising of 43 piroplasmid species, 12 well-defined isolates awaiting formal species description, and two species complexes that possibly mask additional species. The extrapolation of the finding of at least 57 piroplasmid species in only six domestic mammalian groups (cattle, sheep, goat, horse, dog, and cat) allows us to predict that a substantially higher number of piroplasmid parasites than vertebrate host species exist. Accordingly, the infection of a vertebrate host species by multiple piroplasmid species from the same and/or different phylogenetic lineages is commonly observed. Molecular phylogeny using 18S rRNA genes of piroplasmids infecting domestic mammals results in the formation of six clades, which emerge due to an anthropocentric research scope, but not due to a possibly assumed biological priority position. Scrutinizing the topology of inferred trees reveals stunning insights into some evolutionary patterns exhibited by this intriguing group of parasites. Contrary to expectations, diversification of parasite species appears to be dominated by host-parasite cospeciation (Fahrenholz's rule), and, except for piroplasmids that segregate into Clade VI, host switching is rarely observed. When only domestic mammalian hosts are taken into account, Babesia sensu lato (s.l.) parasites of Clades I and II infect only dogs and cats, respectively, Cytauxzoon spp. placed into Clade III only infect cats, Theileria placed into Clade IV exclusively infect horses, wheras Theileria sensu stricto (s.s.) of Clade V infects only cattle and small ruminants. In contrast, Babesia s.s. parasites of Clade VI infect all farm and companion animal species. We outline how the unique ability of transovarial transmission of Babesia s.s. piroplasmids of Clade VI facilitates species diversification by host switching to other host vertebrate species. Finally, a deterioration of sequence fidelity in databases is observed which will likely lead to an increased risk of artifactual research in this area. Possible measures to reverse and/or avoid this threat are discussed.

Degrade to survive: the intricate world of piroplasmid proteases.

Piroplasmids of the genera Babesia, Theileria, and Cytauxzoon are tick-transmitted parasites with a high impact on animals and humans. They have complex life cycles in their definitive arthropod and intermediate vertebrate hosts involving numerous processes, including invasion of, and egress from, host cells, parasite growth, transformation, and migration. Like other parasitic protozoa, piroplasmids are equipped with different types of protease to fulfill many of such essential processes. Blockade of some key proteases, using inhibitors or antibodies, hinders piroplasmid growth, highlighting their potential usefulness in drug therapies and vaccine development. A better understanding of the functional significance of these enzymes will contribute to the development of improved control measures for the devastating animal and human diseases caused by these pathogens.

Role of anti-ß1 adrenergic antibodies from patients with periodontitis in cardiac dysfunction.

BACKGROUND: The presence of serum autoantibodies against ß(1) adrenoreceptors (ß(1)-ARs) in human gingival fibroblast from patients with periodontitis inhibits primary cell-specific growth and induces over-expression of pro-inflammatory mediators. Serum ß(1)-AR autoantibodies from patients with periodontitis react with myocardium and modify cardiac contractility. The relationship between the presence of serum ß(1)-AR autoantibodies and alterations in heart rate variability (HRV) was also studied. METHODS: An enzyme-linked immunosorbent assay (ELISA) using cardiac and gingival fibroblast membranes or synthetic peptides corresponding to the second extracellular loop of human ß(1)-AR was used to detect serum autoantibodies. The HRV was assessed from RR interval files generated from 22:00 to 08:00 hours. The autoantibody effects on contractility were measured on spontaneous rat isolated atria. RESULTS: Circulating autoantibodies from 36 patients with periodontitis and 20 healthy individuals (controls) interacted with fibroblasts, the cardiac surface, and ß(1)-AR synthetic peptides. The distributions of serum antibodies against gingival and myocardium membranes and ß(1)-AR synthetic peptide were 88.8%, 77.7%, and 92.8%, respectively. Moreover, 88.5% of patients with periodontitis whose sera were positive against ß(1)-AR synthetic peptide had decreased HRV. The corresponding affinity-purified anti-ß(1)-AR peptide IgG displayed partial agonist-like activity modifying the isolated atria contractility. CONCLUSION: This manuscript describes that patients with periodontitis showed increased levels of serum IgG with reactive activity against ß(1)-AR. Those patients demonstrated decrease in heart rate, and IgG derived from their sera induced aberrant contractility of heart atrium. We propose that periodontitis increases the risk of cardiovascular diseases, although it increases anti-ß(1)-AR autoantibody that alters myocardial contractility.

International interlaboratory validation of a nested PCR for molecular detection of Babesia bovis and Babesia bigemina, causative agents of bovine babesiosis.

Babesia bovis and B. bigemina are tick-transmitted parasites causing bovine babesiosis, characterized by significant morbidity and mortality leading to economic losses to the livestock industry in tropical and subtropical regions worldwide. Animals that recover from acute infection remain carriers with low parasitemia acting as a source of transmission, and often escape detection. An improved diagnosis of a B. bovis and/or B. bigemina infection of carrier animals is enabled by the availability of detection methods with high sensitivity. To this end, two nested PCR assays targeting the cytochrome b (cytb) genes of B. bovis and B. bigemina (cytb-nPCR), have been recently developed and an increased sensitivity with respect to reference protocols has been shown (Romero-Salas et al., 2016). In this study, the specificity against a panel of hemoparasites that potentially co-occur with B. bovis and B. bigemina was demonstrated to ensure applicability of the cytb-nPCR assays in a wide range of regions where bovine babesiosis is endemic. Furthermore, we compared both reported cytb-nPCR assays with reference nPCR and qPCR protocols for (i) their capability to detect carrier animals in the field, and (ii) their reproducibility when performed in different laboratories by independent operators. We show that, in a panel of bovine field samples (n = 100), the cytb-nPCR assays detected a considerably higher number of 25% B. bovis and 61% B. bigemina-positive animals compared to 7% and 20% B. bovis and 55% and 49% B. bigemina-positive animals when tested by reference nPCR and qPCR protocols, respectively. Cytb-nPCRs were also found superior in the detection of carrier animals when field samples from Africa were analyzed. In addition, both the B. bovis and B. bigemina cytb-nPCR assays were independently validated in a single blinded study in three laboratories. Importantly, no significant differences in the number/percentage of infected animals was observed using cytb-nPCR assays. In summary, the cytb-nPCR assays detected a considerably higher number of chronically infected B. bovis and B. bigemina carrier animals compared to reference nPCR and qPCR protocols, when applied in different epidemiological field situations. Furthermore, a high reproducibility between laboratories could be demonstrated.

Babesia bovis AMA-1, MSA-2c and RAP-1 contain conserved B and T-cell epitopes, which generate neutralizing antibodies and a long-lasting Th1 immune response in vaccinated cattle.

Vaccines against bovine babesiosis must, ideally, induce a humoral immune response characterized by neutralizing antibodies against conserved epitopes and a cellular Th1 immune response. In Babesia bovis, proteins such as AMA-1, MSA-2c, and RAP-1 have been characterized and antibodies against these proteins have shown a neutralizing effect, demonstrating the implication of B and T-cell epitopes in the immune response. There is evidence of the existence of B and T-cell epitopes in these proteins, however, it remains to be defined, the presence of conserved peptides in strains from around the world containing B and T-cell epitopes, and their role in the generation of a long-lasting immunity. The aim in this paper was to identify peptides of Babesia bovis AMA-1, MSA-2c, and RAP-1 that elicit a neutralizing and long-lasting Th1 immune response. Peptides containing B-cell epitopes of AMA-1, MSA-2c and RAP-1, were identified. The immune response generated by each peptide was characterized in cattle. All peptides tested induced antibodies that recognized intraerythrocytic parasites, however, only 5 peptides generated neutralizing antibodies in vitro: P2AMA-1 (6.28%), P3MSA-2c (10.27%), P4MSA-2c (10.42%), P1RAP-1 (32.45%), and P4RAP-1 (36.98%). When these neutralizing antibodies were evaluated as a pool, the inhibition percentage of invasion increased to 52.37%. When the T cellular response was evaluated, two peptides: P3MSA2c and P2AMA1 induced a higher percentage (>70%) of activated CD4 +/CD45RO+ T cells than unstimulated cells. Additionally, both peptides induced the production of gamma interferon (IFN-) in PBMCs from vaccinated cattle after one year proving the implication of a long-lasting Th1 immune response. In conclusion, we identified conserved peptides containing B and T-cell epitopes in antigens of B. bovis that elicit a Th1 immune response and showed evidence that peptides from the same protein elicit different immune responses, which has implication for vaccine development in bovine babesiosis.

Autoantibodies from schizophrenia patients induce cerebral cox-1/iNOS mRNA expression with NO/PGE2/MMP-3 production.

We demonstrated that circulating antibodies from schizophrenia patients, which interact with cerebral M1 muscarinic acetylcholine receptors (M1 mAChRs), trigger production of nitric oxide (NO), prostaglandin E2 (PGE2) and matrix metalloproteinase-3 (MMP-3), and act as inducers of cyclooxygenase-1 (cox-1) and inducible nitric oxide synthase (iNOS) mRNA expression in the rat frontal cortex. The corresponding affinity-purified anti-M1 peptide IgG from schizophrenia patients, while stimulating cerebral M1 mAChRs, increases NOS activity, PGE2 and MMP-3 production associated with iNOS over-activity and mRNA expression. Moreover, PGE2 and MMP-3 production is the result of cox-1 expression and activity. All these effects were inhibited by pirenzepine or haloperidol and mimicked the action of the authentic mAChR agonist. Concurrent analysis of the effects of iNOS, phospholipase C, protein kinase C and calcium/calmodulin inhibition showed that antibody up-regulation of NOS activity, PGE2 and MMP-3 production is under the control of the endogenous NO signalling system. These results provide evidence of the role that cholinergic receptor antibodies play in the development of cerebral inflammation, which shows that an antibody that interacts with cerebral mAChRs can induce expression of pro-inflammatory mediators, and support the participation of an autoimmune process in a particular group of chronic schizophrenia patients.

Highly sensitive nested PCR and rapid immunochromatographic detection of Babesia bovis and Babesia bigemina infection in a cattle herd with acute clinical and fatal cases in Argentina.

Bovine babesiosis is a tick-transmitted haemoparasitic disease caused by Babesia bovis and B. bigemina affecting cattle of tropical and subtropical regions around the world. Pathogens are transmitted by the tick vector Rhipicephalus microplus displaying a widespread distribution in northeastern Argentina. The disease is characterized by significant animal morbidity and mortality resulting in considerable economic loss. In this study, B. bovis and B. bigemina infection was investigated in a cattle herd of 150 adult bovines of pure Braford breed raised in a tick-hyperendemic field using molecular and serum antibody tests. A highly sensitive nested polymerase chain reaction (nPCR) assay targeting a species-specific region of the apocytochrome b gene resulted in direct B. bovis and B. bigemina detection in 27.3% and 54.7% of bovines, respectively. A recently developed immunochromatographic strip test (ICT) based on recombinant forms of spherical body protein 4 and the C-terminal region of rhoptry-associated protein 1 showed that 71.3% and 89.3% of bovines were seropositive for B. bovis and B. bigemina, respectively. The mixed infection rate as observed by direct (19.3%) and indirect detection (65.3%) coincided with those expected, respectively. Importantly, four months after sampling, nine bovines of the studied herd showed clinical signs of bovine babesiosis of which six animals eventually died. Microscopic detection of infected erythrocytes in Giemsa-stained blood smears confirmed B. bovis infection. Our study demonstrates that although animals showed a relatively high and very high rate of immunity against infection with B. bovis (71.3%) and B. bigemina (89.3%) parasites, respectively, clinical cases and fatalities due to the infection with B. bovis were observed. It is proposed that the most adequate control measure in the studied epidemiological situation is to vaccinate animals to prevent losses and/or an outbreak of bovine babesiosis.

IL-1 beta and PGE2 levels are increased in the saliva of children with Langerhans cell histiocytosis.

Langerhans cell histiocytosis (LCH) is a rare disorder mainly of children, whose pathogenesis is still unknown. Some studies have demonstrated that LCH lesions produce different cytokines abnormally that may be relevant to the pathogenesis of the disease. The purpose of this study was to investigate interleukin-1 beta (IL-1 beta) and prostaglandin E2 (PGE(2)) levels in saliva from children with different clinical subtypes of LCH. We studied 29 children with LCH: seven unifocal (Group I), seven multifocal (Group II), 15 multisystemic (Group III) and 12 healthy volunteers (Group IV). Salivary IL-1 beta and PGE(2) levels were significantly higher in LCH than in normal children. A multi-comparison test showed significantly (P < 0.001) higher levels of both IL-1 beta and PGE(2) in saliva from Group III compared with Groups II and I. A significant correlation (r = 0.05) between IL-1 beta and PGE(2) concentrations in saliva from each group was determined. Our findings demonstrated an association between high concentrations of salivary IL-1 beta and PGE(2) and advanced stages of the disease. This allows us to suggest that the abnormal amount of these factors in saliva may serve as a risk marker for disease progression.

Regulation of m1 muscarinic receptors and nNOS mRNA levels by autoantibodies from schizophrenic patients.

In this paper we demonstrate that, circulating antibodies from schizophrenic patients interacting with cerebral M1 muscarinic acetylcholine receptors (M1 mAChRs), can act as an inducer of m1 mAChR-mRNA, and neuronal nitric oxide synthase (nNOS) mRNA gene expression of rat frontal cortex. The different signaling pathways involved in the autoantibody's actions, were characterized. As previously reported serum autoantibodies from schizophrenic patients reacted against neural cells surface inhibiting the binding of the specific mAChR radioligand to rat cerebral frontal cortex membrane. Moreover, by ELISA using M1 synthetic peptide (with identical aminoacid sequence to human M1 mAChR) as coating antigen we demonstrated the reactivity against the second extracellular loop of human cerebral M1 mAChR. The corresponding affinity-purified anti M1 peptide IgG (anti M1 peptide IgG) from schizophrenic patients by stimulation of M1 mAChR exerted an increase in m1 mAChR-mRNA and nNOS-mRNA levels, that significantly correlated with the accumulation of phosphoinositides (IPs) and activation of NOS (alpha = 0.05). All these effects were blunted by pirenzepine and mimicked the action of the authentic agonist. Concurrent analysis of the effects of nNOS, phospholipase C (PLC) and calcium/calmodulin (CaM) inhibition on both, m1 mAChR-mRNA and nNOS-mRNA levels, showing that antibody up-regulation mRNA level is under the control of endogenous nitric oxide (NO) signaling system. On the basis of our results, the activation of M1 mAChR by schizophrenic autoantibody appears to induce nNOS-mRNA expression and reciprocally, the activation of NOS up-regulates m1 mAChR gene expression. These results gave support to the participation of an autoimmune process in a particular group of chronic schizophrenic patients.

Novel insight into the mechanisms involved in the regulation of the m1 muscarinic receptor, iNOS and nNOS mRNA levels.

In this paper we have determined the different signaling pathways involved in M(1) muscarinic acetylcholine receptor (mAChR)-dependent stimulation of m1 mAChRs, neural and inducible isoforms of nitric oxide synthase (nNOS and iNOS)-mRNA gene expression of rat frontal cortex. Carbachol-stimulation of M(1) mAChRs exerts an increase in m1 mAChR-mRNA, activation of phosphoinositide (PI) turnover, translocation of protein kinase C (PKC) and stimulation of NOS activity. Inhibitors of phospholipase C (PLC), calcium/calmodulin and NOS, but not guanylate cyclase, prevent the carbachol-dependent increase of m1 mAChR-mRNA levels. These inhibitors also attenuate the muscarinic receptor-dependent increase in nNOS and iNOS mRNA levels. These results suggest that carbachol-activation of M(1) mAChRs increases m1 mAChR, nNOS and iNOS mRNA levels associated with increased production of nitric oxide (NO). The mechanism appears to occur secondarily to stimulation of PI turnover via PLC activation. This in turn, triggers a cascade reaction involving calcium/calmodulin and PKC, leading to activation of NOS. On the basis of our results, the activation of M(1) mAChRs appears to induce nNOS and iNOS expression and, reciprocally, the activator of NOS up-regulates m1 mAChR gene expression. These results may contribute to a better understanding of the effects and side effects of cholinomimetic treatment in patients with neurodegenerative diseases.

Pro-apoptotic effect of anti-ß1-adrenergic receptor antibodies in periodontitis patients.

An anti-ß(1)-adrenergic antibody from the sera of periodontitis patients (anti-ß(1)-AR IgG) against the second extracellular loop of the human ß(1)-adrenoceptor (ß(1)-AR) has been shown to cause rat atria apoptosis. The anti-ß(1)-AR IgG binds and activates atria ß(1)-AR, increasing the intracellular calcium concentration, which, in turn, activates caspases-3, -8, and -9. The ß(1)-AR and the post-receptor activation of calcium/calmodulin (CaM) lead to increased inducible nitric oxide synthase (iNOS) activity, with an increase in cyclic GMP (cGMP) accumulation as well as increased JNK phosphorylation and cyclic AMP (cAMP) production. We also observed an apoptotic effect of anti-ß(1)-AR IgG, with increased generation of PGE(2). Comparatively, xamoterol, an authentic ß(1)-AR agonist, mimicked the autoantibody effect on rat atria ß(1)-AR apoptosis. Our results suggest that autoantibodies from the sera of periodontitis patients bind and interact with rat atria ß(1)-AR, provoking apoptosis. This implicates a series of modulatory cardiac signaling events that could alter normal heart function and may occur with chronic stimulation of the atria ß(1)-AR, which could lead to heart failure. These results suggest an important link between periodontitis and cardiovascular disease.

Chagasic antibodies induce cardiac COX-2/iNOS mRNA expression with PGE2/NO production.

We demonstrate that serum IgG in chagasic patients interacting with the second extracellular loop of human cardiac M(2) muscarinic acetylcholine receptors (M(2) mAChR) trigger the production of PGE(2) and NO, that in turn induces COX-2/iNOS mRNA expression. An association between serum anti-M(2) peptide IgG, anti-cardiac membrane IgG and PGE(2) levels (p<0.05) in chagasic dysautonomic patients was observed. Thus, we establish that serum anti-mAChR autoantibodies and PGE(2) might be considered as early markers of Chagas' associated dysautonomia. Affinity purified anti-M(2) peptide IgG from chagasic sera, while stimulating myocardial M(2) mAChR, it exerts an increase on PGE(2) generation and NOS activity, as well as COX-2/iNOS isoforms mRNA expression. The expression of these genes is related with phosphoinositides (PIs), cGMP accumulation and PKC activity. Inhibition of these enzymes shows that chagasic autoantibodies up-regulation of COX-2/iNOS mRNA level is under the control of endogenous iNO/cGMP signaling system. These results provide a novel insight into the role that cholinoceptor antibodies play in the development of myocardial inflammation. To our knowledge, there has been no previous report showing that an antibody interacting with heart mAChR can act as expression inducer of proinflammatory mediators.

Cholinoceptor modulation on nitric oxide regulates prostaglandin E(2) and metalloproteinase-3 production in experimentally induced inflammation of rat dental pulp.

The purpose of this study was to investigate the role of muscarinic acetylcholine receptor (mAChR) activity in the regulation of inducible nitric oxide synthase (iNOS) activity, prostaglandin E(2) (PGE(2)), and metalloproteinase-3 (MMP-3) in experimentally induced inflammation of rat incisors dental pulp. Inflammation was induced by application of bacterial lipopolysaccharide (LPS) to the pulp. Extirpated pulp tissue samples were incubated in saline solution until the various experiments were performed. Saline-treated pulp and healthy pulp were used as controls. NOS activity was measured by the production of [U-(14)C]-citrulline from [U-(14)C]-arginine. PGE(2) and MMP-3 production were evaluated by an enzyme-linked immunosorbent assay (ELISA) and cyclooxygenase (cox-1 and cox-2) messenger RNA levels were measured using a reverse-transcriptase polymerase chain reaction by coamplification of target complementary DNA with a single set of primers. The application of LPS to the pulp increased NOS activity, PGE(2), and MMP-3 production associated with iNOS overactivity. Moreover, PGE(2) and MMP-3 production were the result of cox-2 expression. Pilocarpine (5 x 10(-11) mol/L to 5 x 10(-9) mol/L), acting on mAChRs, triggered a negative effect on NOS activity, PGE(2), and MMP-3 production. In control pulp, no action of pilocarpine was observed. Pulpitis changed mAChR conformation, increasing its coupling efficiency to transducing molecules that in turn activate iNOS. The capacity of pilocarpine to prevent iNOS activity, PGE(2), and MMP-3 by acting on mAChR mutation induced by pulpitis might be useful therapeutically as a local treatment.

Role of nitric oxide/cyclic GMP and cyclic AMP in beta3 adrenoceptor-chronotropic response.

In this study we determine different signaling pathways involved in beta(3) adrenoceptor (beta(3)-AR) dependent frequency stimulation in isolated rodent atria. Promiscuous coupling between different G-proteins and beta(3)-AR could explain the multiple functional effects of beta(3)-AR stimulation. We examine the mechanisms and functional consequences of dual adenylate cyclase and guanylate cyclase pathways coupling to beta(3)-AR in isolated rodent atria. The beta(3)-AR selective agonists ZD 7114 and ICI 215001 stimulated in a dose-dependent manner the contraction frequency that significantly correlated with cyclic AMP (cAMP) accumulation. Inhibition of adenylate cyclase shifted the chronotropic effect to the right. On the other hand, the ZD 7114 activity on frequency was enhanced by the inhibition of nitric oxide synthase (NOS) and soluble guanylate cyclase. This countervailing negative chronotropic nitric oxide-cyclic GMP (NO-cGMP) significantly correlated with the increase on NOS activity and cGMP accumulation. Current analysis showed a negative cross talk between cAMP chronotropic and NO-cGMP effects by inhibition of phospholipase C (PLC), calcium/calmodulin (CaM), protein kinase C (PKC), NOS isoforms and Gi-protein on the effects of beta(3)-AR stimulation. RT-PCR detected both eNOS and nNOS in isolated rat atria. NOS isoforms performed independently. Only nNOS participated in limiting the effect of beta(3)-AR stimulation. In eNOS-KO (eNOS-/-) mice the chronotropic effect of beta(3)-AR agonists did not differ from wild type (WT) mice atria, but it was increased by the inhibition of nNOS activity. Our results suggest that the increase in frequency by beta(3)-AR activation on isolated rodent atria is associated to a parallel increases in cAMP. The nNOS-cGMP pathway negatively modulates beta(3)-AR activation. Multiple signal transduction pathways between G-protein and beta(3)-AR may protect myocardium from catecholamine-induced cardiotoxic effects.

Inducible nitric oxide synthase subserves cholinergic vasodilation in retina.

In this paper, we investigate the role of muscarinic acetylcholine receptor (mAChR) activity in the regulation of inducible (i) nitric oxide synthase (iNOS) expression and activity. The signaling pathway involved is also examined. These experiments also provide a link between mAChR activation and the nitric oxide (NO)-dependent regulation of retinal vascular diameter. The diameter of the retinal vessels at a distance of 1 disc diameter from the center of the optic disc was measured in rats using digital retinal photography, and both iNOS-mRNA gene expression and NOS were specifically measured using RT-PCR and [U-(14)C] citrulline assays, respectively. Stimulation of M(1) and M(3) mAChR with carbachol caused an increase in vessel diameter, in iNOS-mRNA levels and in NOS activity in the retina. Aminoguanidine, an inhibitor of iNOS, attenuated all these effects. Inhibitors of phospholipase C (PLC) and protein kinase C (PKC) but not calcium/calmodulin (CaM) prevented the muscarinic-dependent increase in iNOS-mRNA levels. The results obtained suggest that the activation of mAChR increases retinal vessel diameters by increasing the production of nitric oxide (NO) through iNOS activation and iNOS-mRNA gene expression. The mechanism appears to occur secondarily to stimulation of PLC and PKC enzymatic activity.

Correlations between neuronal nitric oxide synthase and muscarinic M3/M1 receptors in the rat retina.

This study determined the different signal pathways involved in M1/M3 muscarinic acetylcholine receptor (mAChR) dependent stimulation of nitric oxide synthase (NOS) activity/cyclic GMP (cGMP) production and nNOS mRNA expression in rat retina. Exposure of the retina to different concentrations of carbachol caused an increase in NOS activity, cGMP production and phosphoinositol (PI) accumulation. The increase in NOS activity and cGMP content was blocked by L-NMMA and ODQ, respectively. Also, phospholipase C (PLC) and calcium/calmodulin (CaM) inhibition prevented the carbachol activation on NOS/cGMP pathways. Both, 4-DAMP and pirenzepine but not AF-DX 116 blocked the increase in NOS and cGMP induced by carbachol. Carbachol-stimulation of M1/M3 mAChR increased nNOS-mRNA levels associated with an increase of endogenous NO and cGMP production. The mechanism appears to occur secondarily to stimulation of PIs turnover via PLC. This triggers a cascade reaction involving CaM and soluble guanylate cyclase leading to NO and cGMP accumulation, that in turn, up regulates nNOS-mRNA gene expression. These results give novel insight into the mechanism involved in the regulation of nNOS-mRNA levels by mAChR activation of retina.