If you choose, you can also include spinach, kale, and beets in your diet on a regular basis. After following this program for three months, have your saliva tested again. Nathan Bryan at the University of Texas Health Science Center helped to develop this product with numerous physiologic tests to ensure the purity and effectiveness of this supplement.

Start working with Cellular Healing to get your questions answered and move towards a rejuvenated you. Loniewski Cellular Healing Blog Next Steps Contact Start with Prayer Book An Appointment eBooks Overview Am I A Candidate? Ten Steps to Healthy Joints.

Nitric Oxide. Patient Services Nitric Oxide. Overview of Nitric Oxide. What is it? An overview of this specific therapy, explained in a way that is comprehensive and concise. How Convenient is it? Get a better understanding of the procedure and what to expect during treatment.

How much does it cost? Understand ahead of time how much this procedure costs. What is Nitric Oxide? These effects may include: Hypertension Angina and Heart Attacks Deep venous thrombosis DVT and blood clots Dementia Erectile dysfunction Chronic obstructive pulmonary disease Constipation Kidney failure Chronic infections The above list is an accurate description of what happens as many people age.

How Convenient is Nitric Oxide Supplementation? How Much Does Nitric Oxide Supplementation Cost? Nitric Oxide Biochemistry basics Habib S, Ali A. The apoptosis signal-regulating kinase 1 ASK1 is regulated by NO [32].

Previous studies indicate that ASK1 is a MAPKKK that activates both JNK and p38 pathways [33]. These results indicate that NO activates ASK1 which subsequently initiates JNK1-dependent cell death. Wild type MEFs were pretreated with 40 µM SP for 30 minutes followed by 0, , and µM DETA-NO for 24 hours.

Percent cell death was measured by LDH release C. Percent LDH release was measured in wild type MEFs pretreated with UO 10 µM followed by 0, , E.

JNK has been reported to inactivate the anti-apoptotic BCL-2 protein, MCL We examined whether the ASK1-JNK1 axis negates MCL-1 as a mechanism for NO-dependent cell death. A widely accepted mechanism for MCL-1 negation following a death stimulus involves proteosomal degradation of the MCL-1 protein [34] , [35].

NO induced a decrease in MCL-1 protein levels upstream of BAX and BAK Figure 9A. The NO-induced decrease in MCL-1 protein was inhibited in cells treated with the proteosomal inhibitor Figure 9B. These results indicate that NO activates ASK1-JNK1 axis to initiate the degradation of MCL The canonical pathway for MCL-1 degradation involves the activation of the BH3-only protein NOXA, which binds MCL-1 and induces its degradation by the proteosome [14] , [36].

MCL-1 is degraded in the absence of NOXA following NO treatment Figure 10A. The E3 ligase, Mule, polyubiquitinates MCL-1 at five lysine residues 5, 40, , and to initiate proteosomal degradation [37]. Mutation of these five critical residues markedly increases the half-life of MCL-1 Figure 10B.

To determine if these five residues are required for NO-induced MCL-1 degradation, MEFs expressing MCL-1 with mutations at all five of these critical residues MCL-1 5K mutant MEFs were exposed to DETA-NO. Surprisingly, the 5K mutant MCL-1 is degraded following treatment with NO Figure 10C.

Additionally, MEFs expressing the 5K mutant die in response to NO Figure 10D. These data indicate that NO induces degradation of MCL-1 through a non-canonical pathway.

Cell lysates were collected at 30 minute intervals for 2 hours and MCL-1 expression was analyzed by Western blot. Tubulin served as a loading control B. MCL-1 5K mutant MEFs were treated with µM DETA-NO for 0, 8, 16 and 24 hours C. Wild type and MCL-1 5K mutant MEFs were treated with 0 and µM DETA-NO for 24 and 48 hours.

Cell death was measured by percent LDH release D. ASK1 and JNK1 are both known to be activated by oxidative stress [38] — [40]. NO can increase mitochondrial oxidative stress by inhibiting cytochrome c oxidase [41].

This could cause the upstream electron transport chain to exist in a more reduced state and enhance superoxide production [5]. Supplementing glucose for galactose reduces glycolysis and forces cells to rely heavily on oxidative phosphorylation for ATP generation and survival.

These results indicate that NO inhibits mitochondrial respiration. PEITC depletes glutathione and generates an increase in intracellular ROS [42]. EUK protected MEFs from PEITC-induced cell death Figure 11B. Additionally, wild type MEFs pre-treated with the peroxynitrite scavengers, uric acid or ebselen, also died at rates similar to controls Figure 11C and 11D.

By contrast, the NO scavenger PTIO decreased DETA-NO-induced cell death Figure 11E. Wild type MEFs were pre-treated with the ROS inhibitor EUK 20 µM followed by 0, , and µM DETA-NO for 24 hours and cell death was measured by LDH release B.

PEITC is known to generate endogenous ROS and was used as a positive control. Wild type MEFs were pre-treated with the peroxynitrite scavengers, uric acid 1 mM and ebselen 10 µM followed by 0, , and µM DETA-NO for 24 hours and cell death was measured by percent LDH release C and D.

Wild type MEFs were pre-treated with the nitric oxide scavenger PTIO 1 mM followed by 0, , and µM DETA-NO for 24 hours and cell death was measured by percent LDH release E. Previous studies have demonstrated that the overexpression of anti-apoptotic BCL-2 family members prevents NO-induced cell death, suggesting that the NO induces cell death through the activation of the intrinsic apoptotic pathway [16].

BAX and BAK are integral members of the intrinsic pathway. Activation of these proteins results in cytochrome c release from the mitochondria and activation of Caspase Our data show that BAX and BAK are activated by NO and that cytochrome c is released from the mitochondria. The combined loss of BAX and BAK, or the individual loss of Caspase-9, completely prevents NO-induced cell death indicating that these proteins are required for this pathway.

Activation of BAX and BAK is dependent on interactions between anti-apoptotic and pro-apoptotic BCL-2 proteins. There are currently two models that attempt to explain how these interactions orchestrate BAX and BAK activation.

Despite differences in these models, both agree that BH3-only proteins are major upstream regulators of BAX and BAK activity. Both models agree that BIM, BID and PUMA are potent activators of apoptosis but through distinct mechanisms.

The indirect activation model views these proteins as potent because they are able to bind and inhibit all of the anti-apoptotic BCL-2 proteins, whereas other BH3-only proteins have selective partners [43]. In this model, the negation of BCL-2 proteins is sufficient to induce BAX and BAK activation.

The indirect activation model proposes that anti-apoptotic BCL-2 members negate the activity of BAX or BAK in healthy cells. In response to a death stimulus, BH3-only proteins such as BIM bind to the anti-apoptotics and displace them from BAX and BAK.

By contrast, the direct activation model proposes that BIM, BID and PUMA are potent because they can directly activate BAX and BAK [15] , [44]. In the present study, we find that cells collectively deficient in Bim and Puma with reduced Bid expression still die in response to NO.

We speculate that a BH3-only protein other than BIM, BID, PUMA, BAD or NOXA negates the anti-apoptotics in response to NO or that NO promotes binding of multiple proteins to anti-apoptotic BCL-2 members, negating their activity. A major mechanism for negating anti-apoptotic BCL-2 protein is through degradation.

For example, MCL-1 is degraded in response to a variety of death stimuli including DNA damage, cytokine withdrawal, and anoxia [20] , [34] , [35] , [45] , [46]. NO induces proteosomal degradation of MCL-1 even in the absence of BAX and BAK, suggesting this is an early event in the pathway.

Previous studies indicate that the BH3-only protein NOXA stimulates MCL-1 degradation upon exposure to a death stimulus. Furthermore, MCL-1 protein is ubiquitinated at five critical lysine residues that are required for degradation.

Surprisingly, NO causes MCL-1 degradation in the absence of NOXA, and a mutant form of MCL-1, that can not be ubiquitinated at the five critical lysine residues, is also degraded.

Collectively, these data indicate that MCL-1 is degraded by a non-canonical mechanism that does not involve NOXA or ubiquitinylation of MCL-1 at five specific lysine residues. The signaling pathways that negate anti-apoptotic BCL-2 proteins to allow BAX and BAK activation are not fully understood.

The JNK and p38 MAPK family members have been implicated as death kinases. Specifically, JNK1 but not JNK2, is implicated in the induction of cell death [21]. JNK can also phosphorylate and inactivate MCL-1 [48] , [49].

Our data indicate that p38α and JNK2 are dispensable in NO induced cell death, but Jnk1 null cells are markedly protected from NO-induced cell death.

Furthermore, NO does not induce MCL-1 protein degradation in the absence of Jnk1. These results indicate that in the absence of Jnk1 , cell survival is due to the maintenance of MCL ASK1 activates the JNK signaling pathway under conditions of high oxidative stress.

ROS oxidize cysteine residues of the Trx protein causing it to dissociate from ASK1 resulting in its activation through autophosphorylation. Indeed, MCL-1 protein levels were maintained in Ask1 deficient cells and these cells did not undergo cell death.

However, we found no evidence that the NO-induced cell death pathway was dependent on oxidative stress. This was surprising since NO can inhibit the mitochondrial respiratory chain and increase oxidative stress [41]. In our experiments, NO was able to block oxidative phosphorylation.

We speculate that NO might cause direct S -nitrosylation of the cysteine residues of Trx thereby liberating ASK1 to induce cell death. These findings have implications for the role of inflammation in cancer progression. Recent data indicate that inflammation can exacerbate cancer progression.

MCL-1 and BCL-X L are up-regulated in many types of cancers. Activated macrophages, which release high levels of NO during inflammation, are capable of destroying neoplastic cells.

However, if BCL-2 proteins are upregulated in tumor cell this would prevent NO-dependent cell death resulting tumor progression.

This would further select tumor cells that have elevated levels of anti-apoptotic BCL-2 proteins and make them resistant to chemotherapy or radiotherapy. MCL-1 5K mutant MEFs and FT were cultured in the above media supplemented with G Cell death was measured using the Cytotoxicity Detection Kit Roche Applied Science according to manufacturer's protocol.

The kit is a colormetric assay based on the measurement of lactate dehydrogenase LDH released by dead cells. Apoptosis was measured using the Annexin V-FITC Apoptosis Detection Kit BD Pharmingen according to manufacturer's protocol.

After treatment, cells were washed twice with cold PBS and resuspended in 1X binding buffer at a concentration of 1×10 6 cells per milliliter. Annexin-V-FITC excitation: nm, emission: was added to the cells and analyzed by flow cytometry. BAX and BAK activation was determined as previously published [23].

Briefly, adherent and non-adherent cells were collected in 1X Cell Dissociation Solution Non-enzymatic Sigma. Cells were fixed in 0. Cells were washed in PBS and analyzed by flow cytometry. Cells were collected in mitochondrial isolation buffer mM Sucrose, 10 mM Tris-HCl pH 7. Cells were then expelled through a guage needle 10 times.

Samples were centrifuged at rpm for 10 minutes. Subsequently, the supernatants were centrifuged at 15,rpm for 20 minutes to pellet the mitochondria. The mitochondrial pellet was solubilized in mitochondrial isolation buffer containing 10X lysis buffer. Phospho-JNK, JNK, phospho-cJun and cJun antibodies were ordered from Cell Signaling.

Briefly, cell lysates were collected in 1X Cell Lysis Buffer 20 mM Tris-HCl pH 7. Membranes were incubated in blocking buffer 1X TBS, 0. The following day, membranes were washed and then incubated in HRP linked anti-mouse or anti-rabbit Cell Signaling for 1 hour.

SuperSignal chemiluminescent substrate Pierce was used to detect protein levels. The pLKO. Constructs were ordered from Open Biosystems with the following hairpin sequences.

Stable cell lines were generated using lentiviral infection using the FT packaging cell line and puromycin selection. The Gateway® Lentiviral Expression Kit Invitrogen was used to overexpress FLAG® tagged BCL-XL.

At its core, the aging process is all about blood flow, Bryan said. Having adequate nitric oxide levels is a key part of maintaining open and flexible blood vessels.

So nitric oxide governs another hidden factor of healthy or not aging—blood pressure. Nor is there any drug that can be used to fix low nitric oxide levels. And there is no drug they can prescribe. And there is the issue of the complicated pathway by which nitrates taken in via food or supplements end up as nitric oxide in the tissues.

It requires a conversion via the right kind of bacteria in the oral microbiome. Proper diet and supplementation can help to address this hidden epidemic, Bryan said. I just got back from a training for chiropractors in Madison, WI.

It will also feature sessions by top suppliers. To close the event, a blockbuster panel of legal, scientific and product development experts will consider the burning questions and future directions of this increasingly vital industry sector.

We will look at formulation questions, market dynamics, claims issues and more.