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NADH: sensor of blood flow need in brain, muscle, and other tissues¹

YASUO IDO^*, KATHERINE CHANG^*, THOMAS A. WOOLSEY and JOSEPH R. WILLIAMSON^*2

^* Department of Pathology,
Department of Neurology and Neurological Surgery, Washington University School of Medicine, St. Louis, Missouri 63110, USA

²Correspondence: Department of Pathology, Box 8118, Washington University School of Medicine, 660 South Euclid Ave., St. Louis, MO 63110-1093, USA. E-mail: jrw{at}pathology.wustl.edu

SPECIFIC AIM

The sensor of blood flow need in conditions as diverse as brain activation, exercise, high altitude, and diabetes mellitus is unknown. Our first aim was to test the hypothesis that accumulation of electrons in cytosolic free NADH, the reduced form of nicotinamide adenine dinucleotide (NAD), functions as this sensor. The rationale for testing this hypothesis was based on the near equilibria between extracellular and intracellular lactate/pyruvate (L/P) and free cytosolic NADH/NAD⁺ ratios; NADH was increased or decreased by injecting lactate or pyruvate, respectively. Our second aim was to identify components of the signaling cascade that mediate increased flows.

PRINCIPAL FINDINGS

1. Increasing L/P increases blood flow in numerous tissues at rest
Bolus injection or brief infusion of lactate increased blood flows in all tissues examined except heart and brain: retina 50%, sciatic nerve 65%, epitrochlearis and gastrocnemius skeletal muscles 30%, soleus muscle 86%, and kidney 40%. Flow increases after lactate infusion for 5 h were retina 45%, sciatic nerve 2.4x, epitrochlearis muscle 3x, diaphragm 17%, and skin 77%. Lactate-augmented flows were prevented when pyruvate was coinjected; injecting pyruvate alone did not affect flow. Similar results were observed with direct application of lactate and/or pyruvate to granulation tissue in a wound-healing chamber model where systemic effects of intravascular injection of lactate and/or pyruvate are obviated.

2. Increasing L/P augments blood flow to contracting muscles
Hind limb skeletal muscle contraction evoked by stimulation of one sciatic nerve (10 Hz for 15 min) increased blood flow 7 to 10x in contracting vs. contralateral resting muscle. Stimulus-evoked flows to muscle were augmented by concurrent infusion of lactate. In contrast, they were prevented by pyruvate infusion, which had no effect on blood flow in contralateral resting muscle. These effects of lactate and pyruvate were abrogated when they were injected together (Fig. 1 ).

View larger version (27K): [in this window] [in a new window]   Figure 1. Effects of lactate and pyruvate vs. saline infusion during 15 min of muscle stimulation on blood flow in resting and contracting adductor magnus muscle. A) In saline controls, blood flow increased 7x in contracting vs. resting muscle. Lactate injection further augmented the elevated blood flows in contracting muscle whereas pyruvate attenuated them; these effects were blunted by coinjecting lactate and pyruvate. Lactate and pyruvate had no effect on blood flow in resting muscle. L-NAME and SODmimic both prevented increased flows in contracting muscle. Flows in the resting muscle were decreased by L-NAME and increased by SODmimic (P<0.05 for both vs. saline controls). Infusions of saline (S), 1 or 2 mmol pyruvate (P) and/or 1 mmol lactate (L)/kg/15 min were initiated at the onset of stimulation. Infusions of SODmimic (1.45 µmol/kg/min) and L-NAME (0.5 µmol/kg/min) were initiated 5 min before muscle stimulation. B, C) Relationships between blood flow in contracting and resting muscles and plasma L/P, pyruvate, and lactate levels after infusion of 1 or 2 mmol pyruvate and/or 1 mmol lactate or saline. Data from individual rats are shown in panel C. Blood flows in contracting muscle correlated positively with plasma L/P (r2=0.57, P<0.0001) and negatively with plasma pyruvate (r2=0.67, P<0.0001) and lactate (r2=0.21, P<0.02). Blood flows in resting muscle did not correlate with plasma L/P, pyruvate, or lactate (r2 0.01, P>0.6 for all 3). D) Blood flow in contracting and resting muscle vs. muscle L/P, pyruvate, and lactate levels. Blood flow did not correlate with muscle L/P, pyruvate, or lactate in contracting muscle (r2<0.05, P>0.25 for all three). In resting muscle, blood flow was weakly (negatively) correlated with muscle L/P (r2<0.14, P=0.042) but not with pyruvate or lactate (r2<0.06, P>0.2 for both). Mean ± SD (A) and mean ± SE (B); SE bars not visible are within the symbols. Numbers of animals are given at the bottom of bars (A) and as subscripts (B). Different from resting muscle at P < 0.05; *different from saline controls at P < 0.05.

3. Increasing L/P augments stimulus-evoked blood flow to whisker barrel cortex
Unilateral whisker stimulation at 7.5 Hz for 5 s increased blood flow 10.5% in contralateral somatosensory whisker barrel cortex vs. ipsilateral unstimulated cortex. Whisker-evoked blood flow increases were doubled by concurrent infusion of lactate and were prevented by pyruvate infusion (which had no effect on blood flow in contralateral resting cortex). As in muscle, these effects of lactate and pyruvate were abrogated when they were coinjected (Fig. 2 ).

View larger version (20K): [in this window] [in a new window]   Figure 2. Blood flow in resting and contralateral somatosensory cortex after 5 s of whisker stimulation at 7.5 Hz. A) Blood flow increased 10% in stimulated vs. resting cortex. Bolus injection of L-lactate (1 mmol/kg in 30 s) 1 min before stimulation further augmented the increased flows in stimulated cortex without affecting resting cortex. Stimulus evoked increases in flow were prevented by a pyruvate bolus (1 mmol/kg) 1 min before stimulation whereas coinjection of lactate and pyruvate had no effect on blood flow in stimulated or resting cortex. Infusion of L-NAME (0.5 µmol/kg/min) or of SODmimic (0.6 µmol/kg/min) started 20 min before stimulation prevented increased flow in stimulated cortex without affecting flow to resting cortex. B) Relationship of blood flows (from panel A) in stimulated and resting cortex to plasma L/P, pyruvate (P), and lactate (L) levels obtained in a separate experiment (S=saline controls). Flows in stimulated cortex parallel increasing plasma L/P independent of pyruvate or lactate levels. Different from resting cortex at P < 0.05; *different from stimulated saline controls at P < 0.05. Other conventions as in Fig. 1 .

4. Blood flow changes correlate with plasma but not tissue L/P
Blood flow in stimulated cerebral cortex paralleled plasma L/P but not lactate and pyruvate levels. Blood flow in contracting skeletal muscle during infusion of saline or lactate and/or pyruvate was strongly and positively correlated with plasma L/P and negatively correlated with plasma lactate and pyruvate levels, but did not correlate with contracting muscle L/P, lactate, or pyruvate. (L/P in extracts of contracting muscle increased 16x vs. resting muscle.) Infusion of lactate or pyruvate during 15 min of muscle stimulation had no effect on blood pCO₂, pO₂, or mean arterial blood pressure; lactate and pyruvate both elevated plasma pH and each increased plasma lactate and pyruvate levels, whereas lactate increased and pyruvate decreased blood flow (Figs. 1 and 2) .

5. Signaling pathways mediating blood flow augmentation by L/P
Injection of a highly selective superoxide dismutase (SOD)_mimic (to block vascular effects of O₂^-) or of L-NAME [a nonselective inhibitor of nitric oxide synthase (NOS) to block ^•NO vasodilation] prevented increased blood flows in working cortex and muscle. They also blocked L/P-augmented flow in skin chambers.

CONCLUSIONS AND SIGNIFICANCE

For more than a century it has been known that increased blood flow with muscle and neural work is coupled to increased energy metabolism and O₂ consumption. Here we show for a wide range of resting and working tissues that blood flow need is sensed by a common mechanism that is not necessarily coupled to increased energy need or metabolism, O₂ consumption, or glycolysis. The observation that blood flow is augmented when L/P ratios are raised and is attenuated when they are lowered (Figs. 1 and 2) supports the conclusion that cytosolic free NADH senses blood flow need (Fig. 3 ). We have understood for more than half a century that the cofactor NAD is the major carrier of electrons (and protons) from fuels for energy metabolism. It is remarkable to discover only now that free cytosolic NAD also functions as the sensor to signal increased blood flow (Fig. 3) .

View larger version (39K): [in this window] [in a new window]   Figure 3. A) Generic cell (i.e., skeletal muscle, neural, and glial cells, as well as vascular smooth muscle and endothelial cells) and nearby blood vessel showing central position of free cytosolic NAD in energy metabolism and blood flow signaling. Since intra- and extracellular L/P are in near equilibrium with NADH/NAD+ under steady-state conditions, changes in extracellular L/P and intracellular L/P in working cells cause corresponding changes in NADH in vascular smooth muscle and endothelial cells. Pyruvate and lactate diffusing from parenchymal cells and from plasma can pass through the vessel wall between and through smooth muscle and endothelial cells. B) Proposed signaling pathway. Neural activity and muscle contraction are fueled by hydrolysis of ATP to ADP. Higher ADP/ATP accelerates glycolysis and transfer of electrons and protons to NAD+ reducing it to NADH faster than electrons can be used for ATP synthesis. Excess electrons carried by NADH fuel redox signaling pathways to increase blood flow by augmenting O2- production and •NO synthesis. (See text for details.)

Increased glycolysis during work and hypoxia accelerates production of pyruvate and transfer of electrons and protons from glucose to NAD⁺ faster than they can be used for ATP synthesis by oxidative phosphorylation (OP) in mitochondria. The advantage of this high rate of glycolysis is that ATP can be synthesized up to 2x faster by substrate phosphorylation (SP) in the cytoplasm than by OP, albeit the yield of ATP (per mol of glucose) from SP is much less than from OP. The high rate of ATP synthesis by SP with increased glycolysis is short lived, however, since glycolysis is inhibited by increases in NADH and by accumulation of lactate (formed by reduction of excess pyruvate to lactate coupled to oxidation of NADH to NAD⁺ by L-DH) under aerobic and hypoxic conditions. Increasing blood flow removes lactate and other products of energy metabolism and augments delivery of fuels and O₂ for energy metabolism and of pyruvate (which serves as a sink to remove more electrons and protons from NADH as well as fuel for energy metabolism).

Excess electrons accumulating in free cytosolic NADH from whatever cause (increased glycolysis with work, elevated extracellular L/P, increased oxidation of sorbitol in diabetes, and hypoxia) fuel redox signaling pathways (coupled to reoxidation NADH to NAD⁺). The observations that pyruvate, an SOD_mimic, and an inhibitor of nitric oxide synthase each prevent lactate and work-induced increased blood flows supports the conclusion that excess electrons in NADH augment production of O₂^- and ^•NO, which mediate the increase in blood flow. Accumulation of electrons in NADH also augments de novo synthesis of diacylglycerol to activate PKC-mediated signaling pathways including PKC-mediated increased glucose transport.

The signaling cascade that mediates work-evoked increases in tissue blood flow is normally initiated by accumulation of electrons in NADH in working parenchymal cells, e.g., contracting muscle cells. The present experiments demonstrate that injection of lactate to increase extracellular (plasma) L/P also increased blood flows in both resting and working cells. These observations, together with the finding that increased blood flows evoked in contracting muscle by work and by lactate injection correlate with plasma (but not contracting skeletal muscle) L/P, indicate that the redox signaling cascade can be initiated in vascular smooth muscle and endothelial cells. Several lines of evidence suggest that chronic activation of these redox-mediated signaling pathways may stimulate new vessel growth to meet increased blood flow need over the longer term, such as with exercise, hypoxia, and diabetic retinopathy.

We observed a wide and seemingly appropriate range of tissue-specific responses or ‘thresholds’ to increases in plasma L/P. In the brain, elevation of plasma L/P increased flow only during work; in skeletal muscle elevation of plasma L/P increased flow in resting and working muscle, but the threshold was markedly decreased by work. These tissue differences likely relate to a number of tissue specific characteristics such as basal metabolism, activity of monocarboxylate transporters, capacity to reoxidize NADH, and content of SOD and NOS.

Our observations identify a novel central role for NAD in sensing blood flow need and augmenting flow that is elegant in its simplicity.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0652fje ; to cite this article, use FASEB J. (April 6, 2001) 10.1096/fj.00-0652fje

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