Vascular Smooth Muscle NO Exposure from Intraerythrocytic SNOHb: A Mathematical Model

Vascular Smooth Muscle NO Exposure from Intraerythrocytic SNOHb: A Mathematical Model Kejing

1 Chen ,

Roland N.

2 Pittman ,

and Aleksander S.

1 Popel

1Department

2Department

of Biomedical Engineering, School of Medicine, Johns Hopkins University, Baltimore, MD 21205; of Physiology, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298

Nitric oxide (NO) is a potent vasodilator on the microvasculature. We have previously constructed computational models based on biochemical pathway analysis of different nitric oxide synthase isoforms and found a large discrepancy between our predictions and perivascular NO measurements, suggesting the existence of non-enzymatic sources of NO. One potential source is red blood cells (RBCs), which have been hypothesized to preserve NO bioactivity. S-nitrosohemoglobin (SNOHb) in RBCs has been put forward as a major contributor to NO-induced hypoxic vasodilation; however, the amount of NO delivered by intraerythrocytic SNOHb to smooth muscle has not been quantified experimentally or calculated using mathematical models. In the present study, we have formulated a multicellular computational model to quantify NO exposure in arteriolar smooth muscle when the NO released by SNOHb is the sole NO source in the vasculature. Our calculations show an NO exposure of ~6 pM in the smooth muscle region. This amount is far below the measured values for the perivascular NO concentration, which are generally reported as several hundred nM, and it does not account for the large discrepancy that we encountered. We found that the amount of NO delivered by SNOHb to smooth muscle strongly depends on the SNOHb concentration and half-life, which further determine the rate of NO release, as well as on the membrane permeability of RBC to NO. In conclusion, our mathematical model predicts that picomolar amounts of NO can be delivered to the vascular smooth muscle by intraerythrocytic SNOHb; how this amount of NO alone might induce hypoxic vasodilation requires further investigation.

NO concentration vs. membrane permeability

Governing equation and model parameters

Abstract

∂CNO = DNO ∇2CNO + RNO ∂t • • •

Model Parameters. The radius of each layer, except r1, represents the distance from the center of the lumen to the outer boundary of the layer Radius RBC (r1) RBC-rich layer (r2) RBC-free layer (r3) Endothelium and interstitial space (r4) Smooth muscle layer (r5) Non-perfused tissue (r6) Perfused tissue (r7)

We consider the two-dimensional crosssection of an arteriole and its surrounding tissues The governing equation is applied to all layers We consider a steady state of the system: ∂CNO = 0 ∂t

• • • • •

The effect of convection is ignored NO enzymatic NO production The SNOHb concentration does not change with time The exported NO bioactivity from RBC is in the form of nitrogen monoxide (free NO) NO release is through a facilitated through cell mechanism, rather than a uniform reaction within an RBC; the surface release is S = Q ⋅V (V: volume; A: NO

NO

A

area), where the volumetric production rate Q is determined by Q = C ⋅ lnt 2 NO

NO

4 15 17 18 24 30 50

µm µm µm µm µm µm µm

RBC membrane thickness (rmem) 0.0078 µm 450 µm/s RBC membrane permeability (PRBC) 3.51 µm2/s NO diffusion coefficient in membr. (Dmem) 3300 µm2/s Extracellular NO diffusion coefficient (Dext) 880 µm2/s Intracellular NO diffusion coefficient (Dint) 18 µM-1·s-1 Hb-NO reaction rate (kHb) Intracellular Hb concentration (CHb) 20 mM 0.05 µM-1·s-1 Reaction rate with sGC (ksGC) Oxygen concentration (CO2) 0 – 100 µM -6 -2 9.6×10 µM ·s-1 Reaction rate with O2 (kO2) SNOHb concentration (CSNOHb) 1333 nM 240 s SNOHb half-life time (t1/2) 5.2 ×10-18 µmol·µm-2·s-1 NO surface release rate (SNO) NO consumption rate by perfused tissue (Kcap) 12.4 s-1 Hematocrit 45% N/A

NO delivered by SNOHb is strongly dependent on the intrinsic resistance of RBC membrane to NO

Influence of the RBC-free zone smooth muscle

SNOHb

1/ 2

NO delivered by SNOHb

Background

smooth muscle

• Reported values of perivascular NO concentrations are generally in the range of several hundred nM • Our biochemical pathway analyses have shown that NO synthase (NOS)derived NO cannot account for the large concentration of NO measured in the perivascular region, suggesting the existence of non-enzymatic NO sources in the vasculature

NO concentration, pM

12

• S-nitrosohemoglobin (SNOHb) hypothesis suggests a novel endocrine signaling pathway: NO binds to T state hemoglobin to form Fe2+NO, then is transferred from the ferrous iron group to the cysteine residue at position 93 on the β chains. Upon hypoxia, the NO to the thiols of the anion exchange protein on the membrane, or to glutathione. NO is then further exported from the erythrocytes and induces vasodilation

8

without cell-free zone with cell-free zone

4 smooth muscle

0 0

smooth muscle

5

10

15

20

25

30

R, µm

Effect of the facilitated membrane release mechanism

• Little quantitative knowledge of the transport and distribution of SNOHbreleased NO is known. Is SNOHb-delivered NO sufficient to induce the vascular smooth muscle relaxation through the known NO-sGC-cGMP pathway?

smooth muscle 0.012

NO concentration, pM

• In the present study, we have used a mathematical model to analyze the NO released through the RBC pathway and predict the NO concentration in the vascular smooth muscle cells, and compared that to that of vascular and perivascular enzymatic sources

12

Model formulation NO concentration, pM

10

0.008

smooth muscle

0.004

0.000 smooth muscle

8

0

5

10

15

20

25

30

R, µM

6

NO delivery to smooth muscle is three order of magnitude lower if the NO release is uniform inside RBCs, rather than a facilitated surface release

4 2 0 0

5

10

15

20

25

Further simulations: different O2 and hematocrit levels do not significantly affect the NO delivery by SNOHb

30

R, µm

1. ~6 pM NO present in smooth muscle 2. Smooth muscle NO exposure is not strongly dependent on the positioning of RBCs

Conclusions

(1) the intraluminal layer containing discrete RBCs and plasma. The RBC is further divided into two sub-regions: a thin membrane and the homogeneous intraerythrocytic hemoglobin (2) endothelium and interstitial space (3) smooth muscle cells (4) non-perfused tissue containing nerve fibers and parenchymal cells (5) tissue perfused by capillaries

700 600 500 400 300 200 100 0 0

40

80

SNOHb concentration, µM

120

NO concentration in smooth muscle, pM

An arteriole and its surrounding tissues. It consists of five layers:

NO concentraion in smooth muscle, pM

NO concentration vs. SNOHb concentration and half-life

• We predicted that ~6 pM NO delivered by intraerythrocytic SNOHb to the vascular wall; whether this amount of NO alone can induce hypoxic vasodilation requires further investigation • The amount of NO present in the smooth muscle depends strongly on the facilitated membrane transport, membrane resistance to NO diffusion, and the physiological level and half-life time of SNOHb

60

• The predicted values of vascular smooth muscle NO concentration does not account for the discrepancy that we have encountered

40

20

Selected references

0 0

600

1200

1800

2400

t1/2, s

NO delivery is strongly dependent on [SNOHb] and t1/2, which further determine NO release rate

1. El-Farra, N. H., Christofides, P. D. and Liao, J. C. (2003) Analysis of nitric oxide consumption by erythrocytes in blood vessels using a distributed multicellular model. Ann Biomed Eng 2. Rogers, S. C., Khalatbari, A., Gapper, P. W., et al (2005) Detection of human red blood cell-bound nitric oxide. J Biol Chem 3. Chen and Popel. (2006) Theoretical analysis of biochemical pathways of nitric oxide release from vascular endothelial cells. Free Radic Biol Med. 4. Chen and Popel. (2007) Vascular and Perivascular NO Release and Transport: Biochemical Pathways of neuronal nitric oxide synthase (NOS1) and endothelial nitric oxide synthase (NOS3). Free Radic Biol Med.