Friday, October 1, 2010

Be Tall with Guanyl Cyclase

Earlier in the article on natriuretic peptides, specifically C-type natruiretic peptide, we learned of a composition for increasing body height involving Guanyl Cyclase and C-type Natruiretic peptide.  How can Guanyl Cyclase be used to increase height?  Now Nitric Oxide leads to a strong increase in Guanyl Cyclase activity.  Nitric Oxide has negative effects as well like DNA Damage(though you can probably mitigate this with folic acid and possibly folinic acid).  Nitric Oxide deficiency has been found to reduce chondrocyte growth(Nitric Oxide is created by breaking down Arginine).

What can Guanyl Cyclase(and as a result Nitric Oxide) do to make us be tall?  

A pulsing electric field (PEF) increases human chondrocyte proliferation through a transduction pathway involving nitric oxide signaling. 

"A potential treatment modality for joint pain due to cartilage degradation is electromagnetic fields (EMF) that can be delivered, noninvasively, to chondrocytes buried within cartilage. A pulsed EMF in clinical use for recalcitrant bone fracture healing has been modified to be delivered as a pulsed electric field (PEF) through capacitive coupling[meaning there's a capacitor involved]. It was the objective of this study to determine whether the PEF signal could have a direct effect on chondrocytes in vitro. This study shows that a 30-min PEF treatment can increase DNA content of chondrocyte monolayer by approximately 150% at 72 h poststimulus. Studies intended to explore the biological mechanism showed this PEF signal increased nitric oxide measured in culture medium and cGMP measured in cell extract within the 30-min exposure period[cGMP is another thing that has been suggested that may increase height, Guanyl Cyclase is needed to catalyze cGMP]. Increasing calcium in the culture media or adding the calcium ionophore A23187, without PEF treatment, also significantly increased short-term nitric oxide production. The inhibitor W7, which blocks calcium/calmodulin, prevented the PEF-stimulated increase in both nitric oxide and cGMP. The inhibitor L-NAME, which blocks nitric oxide synthase, prevented the PEF-stimulated increase in nitric oxide, cGMP, and DNA content. An inhibitor of guanylate cyclase (LY83583) blocked the PEF-stimulated increase in cGMP and DNA content. A nitric oxide donor, when present for only 30 min, increased DNA content 72 h later. Taken together, these results suggest the transduction pathway for PEF-stimulated chondrocyte proliferation involves nitric oxide and the production of nitric oxide may be the result of a cascade that involves calcium, calmodulin, and cGMP production." 

So we don't necessarily need Pulsed Electric Fields to replicated the results just calcium and nitric oxide(and functional Guanyl Cyclase).   The advantage of PEMF is that it can be used on cell types deep within the body.  It's a way of more directly mechanically loading deeper parts like bone marrow stem cells.  The increase in cell proliferation can be mimicked by IGF-1.  The majority of PEMF's cellular proliferation effects seem to be involving the Nitric Oxide pathway which allows for numerous alternatives, the ability of PEMF to induce chondrogenic differentiation will determine whether PEMF is just a supplementary way to increase height or a height increase method all on it's own.

Intact kinase homology domain of natriuretic peptide receptor-B is essential for skeletal development.

"Natriuretic peptide receptor-B (NPR-B, GC-B in rodents; gene name NPR2) is a guanylyl cyclase-coupled receptor that mediates the effect of C-type natriuretic peptide. Homozygous mutations in human NPR-B cause acromesomelic dysplasia, type Maroteaux (OMIM 602875), an autosomal recessive skeletal dysplasia. NPR-B has an intracellular kinase homology domain (KHD), which has no kinase activity, and its functional significance in vivo is currently unknown.
 A 28-yr-old Japanese male presented with marked short stature (118.5 cm, -9.3 sd). His limbs showed marked shortening in the middle and distal segments. His parents had relatively short stature with height z-scores of -2.75 and -0.98 (his father and mother, respectively). Direct sequencing of coding region of the NPR2 gene of the family was performed. The mutant receptor activity was investigated by saturation binding assay and cGMP measurement. Additionally, interaction between the mutant and wild type allele was investigated by the titration experiments.
We identified a novel missense mutation L658F in KHD of NPR-B in homozygous and heterozygous states in the patient and his parents, respectively. The mutation conferred normal binding affinity for C-type natriuretic peptide but no discernible ligand-induced cGMP production. Furthermore, L658F mutant impaired wild-type NPR-B-mediated cGMP production in a dose-dependent manner, suggesting that short stature found in L658F heterozygote can be caused by its dominant-negative effect.
This study provides the first evidence that intact KHD of NPR-B is essential for skeletal development."

Guanyl cyclase is essential for cGMP production. 

Natriuretic Peptides, Their Receptors, and Cyclic Guanosine Monophosphate-Dependent Signaling Functions  

"Natriuretic peptides are a family of structurally related but genetically distinct hormones/paracrine factors that regulate blood volume, blood pressure, ventricular hypertrophy, pulmonary hypertension, fat metabolism, and long bone growth. The mammalian members are atrial natriuretic peptide, B-type natriuretic peptide, C-type natriuretic peptide, and possibly osteocrin/musclin. Three single membrane-spanning natriuretic peptide receptors (NPRs) have been identified. Two, NPR-A/GC-A/NPR1 and NPR-B/GC-B/NPR2, are transmembrane guanylyl cyclases, enzymes that catalyze the synthesis of cGMP. One, NPR-C/NPR3, lacks intrinsic enzymatic activity and controls the local concentrations of natriuretic peptides through constitutive receptor-mediated internalization and degradation. Single allele-inactivating mutations in the promoter of human NPR-A are associated with hypertension and heart failure, whereas homozygous inactivating mutations in human NPR-B cause a form of short-limbed dwarfism known as acromesomelic dysplasia type Maroteaux. The physiological effects of natriuretic peptides are elicited through three classes of cGMP binding proteins: cGMP-dependent protein kinases, cGMP-regulated phosphodiesterases, and cyclic nucleotide-gated ion channels[the third is probably way they were affected by Electric Fields]. In this comprehensive review, the structure, function, regulation, and biological consequences of natriuretic peptides and their associated signaling proteins are described." 

"CNP has been documented to regulate endochondral ossification through the promotion of chondrocyte proliferation and hypertrophy in the growth plate. We are the first to demonstrate a role for CNP in the early stages of chondrogenesis. We show that as cells become chondrogenic, they dramatically increase expression of the CNP receptor Npr2, providing a mechanism for CNP signaling. Compared with the strong increase in Npr2 levels, the biological effects of CNP treatment appear somewhat modest. One potential explanation is that the simultaneous up-regulation of Npr3, encoding the decoy receptor, limits cellular responses to CNP. These data suggest that the biological activities of CNP in cartilage are tightly regulated, in agreement with our recent observation that CNP induces Npr3 expression at later stages of chondrocyte differentiation.  

We demonstrate that CNP treatment stimulates GAG accumulation[chondroitin is an example of a GAG, so CNP treatment would make Chondroitin treatment more effective]. Furthermore, CNP treatment stimulates cellular condensations as demonstrated by increased PNA staining and mRNA levels of the cellular adhesion molecule N-cadherin. It is interesting that in response to CNP treatment, micromass cultures display a 60% increase in Alcian blue staining (after normalization to protein levels) but only a 30% increase in the number of condensations. These data suggest that the increase in Alcian blue staining is in part due to increased synthesis of GAGs per condensation and not just to an increase in the number of condensations." 

Nitric Oxide stimulates Guanyl Cyclase which is needed to catalyze cGMP which is needed for the effects of C-type Natuiritic Peptides to work... And CNPs make Chondroitin more effective plus encourage chondrocyte proliferation.

"Condensing mesenchymal cells express the cell adhesion molecules N-cadherin and N-CAM and the disaccharide galactosidase(ß1,3) N-acetyl galactosamine, which can be visualized by staining with the lectin peanut agglutinin (PNA). The mesenchymal cells within the condensations are stimulated to differentiate into chondrocytes, the cell type of cartilage"<-Condensing is an important part of MSCs to condense into chondrocytes.

"CNP is an important regulator of chondrogenesis through the regulation of mesenchymal condensations and matrix biosynthesis."<-Matrix biosynthesis is likely to involve Chondroitin and Glucosamine.  Thus Chondroitin and Glucosamine may help increase height.

"stimulation of chondrogenesis by CNP occurs independently of changes in Sox9 expression or Sox9 activity. "<-like by Chondroitin and Glucosamine.

"xylosyltransferases I and II are necessary for the synthesis of chondroitin sulfate. mRNA levels for xylosyltransferase I increased strongly during chondrogenesis. CNP treatment resulted in a significant increase in expression of xylosyltransferase I."<-CNP treatment strongly increases chondroitin sulfate synthesis.  If the main action of CNP to grow taller is through GAGs then taking GAGs themselves should help you grow taller.

"We demonstrate in a tibia organ culture system that pharmacological inhibition of p38 blocks the anabolic effects of CNP. We further show that CNP stimulates endochondral bone growth largely through expansion of the hypertrophic zone of the growth plate, while delaying mineralization. Both effects are reversed by p38 inhibition. We also performed Affymetrix microarray analyses on micro-dissected tibiae to identify CNP target genes. These studies confirmed that hypertrophic chondrocytes are the main targets of CNP signaling in the growth plate, since many more genes were regulated by CNP in this zone than in the others. While CNP receptors are expressed at similar levels in all three zones, cGMP-dependent kinases I and II, important transducers of CNP signaling, are expressed at much higher levels in hypertrophic cells than in other areas of the tibia, providing a potential explanation for the spatial distribution of CNP effects. In addition, our data show that CNP induces the expression of NPR3, a decoy receptor for natriuretic peptides, suggesting the existence of a feedback loop to limit CNP signaling. Finally, detailed analyses of our microarray data showed that CNP regulates numerous genes involved in BMP signaling and cell adhesion." 

cGMP, CNPs, Guanyl Cyclase, and Nitric Oxide are the basis for the development of a lot of new height increase formulas and give us a new basis to study.  Like IGF-1, they would help increase the peak size of hypertrophic chondrocytes so likely would only help if applied at a sustained level throughout development(though they do increase chondrocyte proliferation too).  

"The natriuretic peptide family consists of three structurally related endogenous ligands: atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). The biological actions of natriuretic peptides are thought to be mediated through the activation of two guanylyl cyclase (GC)-coupled receptor subtypes (GC-A and GC-B). In this study, we examined the effects of ANP and CNP, which are endogenous ligands for GC-A and GC-B, respectively, on bone growth using an organ culture of fetal mouse tibias, an in vitro model of endochondral ossification. CNP increased the cGMP production much more potently than ANP, thereby resulting in an increase in the total longitudinal bone length. Histological examination revealed an increase in the height of the proliferative and hypertrophic chondrocyte zones in fetal mouse tibias treated with CNP. The natriuretic peptide stimulation of bone growth, which was mimicked by 8-bromo-cGMP, was inhibited by HS-142-1, a non-peptide GC-coupled natriuretic peptide receptor antagonist. The spontaneous increase in the total longitudinal bone growth and cGMP production was also inhibited significantly by HS-142-1. CNP mRNA was expressed abundantly in fetal mouse tibias, where no significant amounts of ANP and BNP mRNAs were detected. A considerable amount of GC-B mRNA was present in fetal mouse tibias. This study suggests the physiologic significance of the CNP/GC-B pathway in the process of endochondral ossification."

CNP increases height by increasing peak chondrocyte hypertrophy and possibly chondrocyte proliferation.  Whether CNP can induce chondrocyte differentiation is unclear. 

"natriuretic peptides can regulate the proliferation and differentiation of osteoblasts, chondrocytes, and osteoclasts"<-But no indication that it can do this to stem cells.

"Both ANP and CNP increased the total longitudinal length of mouse tibias significantly and dose-dependently compared with vehicle-treated groups. Treatment of mouse tibias with 10−7 m CNP for 6 days produced an ∼25% increase in the total longitudinal bone length compared with vehicle-treated groups"<-that's pretty dramatic.

"Treatment of mouse tibias with 10−7 to 10−4 m 8-Br-cGMP for 6 days increased the total bone growth significantly and dose-dependently"<-cGMP can work as well as CNP

"strongly suggest that natriuretic peptide activation of chondrogenesis is mediated primarily by GC-B"<-Guanyl Cyclase B is more important for increasing height than A.

Chronically elevated plasma C-type natriuretic peptide level stimulates skeletal growth in transgenic mice.

"C-type natriuretic peptide (CNP) plays a critical role in endochondral ossification through guanylyl cyclase-B (GC-B), a natriuretic peptide receptor subtype. Cartilage-specific overexpression of CNP enhances skeletal growth and rescues the dwarfism in a transgenic achondroplasia model with constitutive active mutation of fibroblast growth factor receptor-3. Due to the high clearance of CNP, maintaining a high concentration is technically difficult. However, to model high blood CNP concentration, we established a liver-targeted CNP-overexpressing transgenic mouse (SAP-CNP tgm). SAP-CNP tgm exhibited skeletal overgrowth in proportion to the blood CNP concentration and revealed phenotypes of systemic stimulation of cartilage bones, including limbs, paws, costal bones, spine, and skull. Furthermore, in SAP-CNP tgm, the size of the foramen magnum, the insufficient formation of which results in cervico-medullary compression in achondroplasia, also showed significant increase. CNP primarily activates GC-B, but under high concentrations it cross-reacts with guanylyl cyclase-A (GC-A), a natriuretic peptide receptor subtype of atrial natriuretic peptides (ANP) and brain natriuretic peptides (BNP). Although activation of GC-A could alter cardiovascular homeostasis, leading to hypotension and heart weight reduction, the skeletal overgrowth phenotype in the line of SAP-CNP tgm with mild overexpression of CNP did not accompany decrease of systolic blood pressure or heart weight."

"SAP-CNP tgm exhibited systemic elongation of cartilage bones such as limb, paw, and spine. During postweaning development, increased body length was observed in proportion to plasma CNP level"

CNP increased the length of the non-long bones more than long bones.

"The costal growth plates of SAP-CNP tgm exhibited 63% higher synthesis of proteoglycan synthesis than that of the wild-type littermates"

CNP increased the thickness of all layers including resting zone.

A human skeletal overgrowth mutation increases maximal velocity and blocks desensitization of guanylyl cyclase-B.

"C-type natriuretic peptide (CNP) increases long bone growth by stimulating guanylyl cyclase (GC)-B/NPR-B/NPR2. Recently, a Val to Met missense mutation at position 883 in the catalytic domain of GC-B was identified in humans with increased blood cGMP levels that cause abnormally long bones. Here, we determined how this mutation activates GC-B. In the absence of CNP, cGMP levels in cells expressing V883M-GC-B were increased more than 20 fold compared to cells expressing wild-type (WT)-GC-B, and the addition of CNP only further increased cGMP levels 2-fold. In the absence of CNP, maximal enzymatic activity (Vmax) of V883M-GC-B was increased 15-fold compared to WT-GC-B but the affinity of the enzymes for substrate as revealed by the Michaelis constant (Km) was unaffected. Surprisingly, CNP decreased the Km of V883M-GC-B 10-fold in a concentration-dependent manner without increasing Vmax. Unlike the WT enzyme the Km reduction of V883M-GC-B did not require ATP. Unexpectedly, V883M-GC-B, but not WT-GC-B, failed to inactivate with time. Phosphorylation elevated but was not required for the activity increase associated with the mutation because the Val to Met substitution also activated a GC-B mutant lacking all known phosphorylation sites. We conclude that the V883M mutation increases maximal velocity in the absence of CNP, eliminates the requirement for ATP in the CNP-dependent Km reduction, and disrupts the normal inactivation process."

"CNP binding increases GC-B activity by two mechanisms. It increases the maximal rate of cGMP production called maximal velocity (Vmax) and it also increases the affinity of the enzyme for GTP that is observed as a reduction in the Michaelis constant — the GTP concentration required to reach half the Vmax."

"ATP is required for the CNP-dependent reduction in the Km of GC-B"

"ATP increases GC-B activity by providing the phosphate that is added to the serine and threonine residues on the enzyme that is necessary for activation by CNP"

"markers of chondrocyte differentiation (PTH1R, Ihh, and Runx2) were expressed in prehypertrophic to hypertrophic chondrocytes in similar patterns in both SAP-CNP tgm and wild-type littermates"

"BNP transgenic mice exhibit skeletal overgrowth via activating GC-B without body weight change"

"The femur, skull, and spine (L2-4) measurements were longer than that of the wild-type littermates, suggesting that chronically elevated plasma CNP levels stimulate cartilage bones systemically"

"CNP selectively affects only cartilage relating to endochondral ossification."

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