MICROVASCULAR

RESEARCH

13,21I-224 (1977)

Effects of Altered Carbon Dioxide Hemoglobin Oxygenation in Hamster M icrovessels’ ROLAND N. PITTMAN

AND

BRIAN

Tension Cheek

on Pouch

R. DULING’

Department of Physiology, Virginia Commonwealth University, MedicaI College of Virginia, Richmond, Virginia 23298 and Department of Physiology, School of Medicine, University of Virginia, Charlottesville, Virginia 22901 Received February 6,1976 Simultaneous determinations of percentage Hb02 and PO* were made at various sites moving progressively downstream from large to small arterioles in the suffused hamster cheek pouch microcirculation. A new video densitometric technique was employed to measure intravascular percentage oxyhemoglobin (% HbO,), and oxygen tension (PO,) was determined polarographically with oxygen microcathodes. Microvascular diameter and red cell velocity were also measured. The above measurements were made under conditions of sustained maximal vasodilation with with 1O-4 M adenosine in the suffusion solution. The pairs of values for % HbO, andPO1ateachofthemeasurementsiteswereused toconstructapparentinvivoHb0, saturation curves. Lateral shifts of the saturation curve were produced by altering the carbon dioxide tension (X0,) of the suffusion solution from 32 to IO mm Hg, and from 32 to 75 mm Hg. Hill’s equation for the HbOz saturation curves was used to analyze the ‘A HbO, and PO, data pairs. For each value of suffusion solution PC02, PsOwas determined and yielded values of 25 f 3,30 rt 3, and 41 +_4 mm Hg for solution PC02’s of IO, 32, and 75 mm Hg, respectively. The in vivo values are qualitatively in agreement with predictions from published Hb02 saturation curves, although the predicted P5, during suffusion with the hypocapnic solution (X0, = 10 mm Hg) was somewhat higher than expected. The sustained vasodilation produced by adenosine did not in itself lead to a statistically significant change in PsO between normal and dilated arterioles. A pronounced longitudinal decrease in PO, from large to small arterioles with an accompanying fall in % HbO, was observed in all experiments. This longitudinal gradient in arteriolar oxygen content confirmsand extends the previous reports of a progressive decline in precapiIlaryPOz. In addition, evidence is presented which suggests the existence of a precapillary longitudinal gradient in PCOZ.

INTRODUCTION Alteration of blood carbon dioxide tension (PCOJ elicits a dual response in terms of microvascular oxygen delivery. The vasodilator effect of increased blood PCO, causes increased blood flow and the increased PCOZ decreases the affinity of red cell 1 This investigation was supported in part by American Heart Association grant 71993, USPHS grant HL 12792,USPHS grant HL 18292,and a grant from the A. D. Williams Foundation. A preliminary report of this work appeared as an abstract in The Physiologist 18,353, 1975. ‘This work was done during tenure as an Established Investigator of the American Heart Association. Copyright 0 1977 by Academic Press, Inc. 211 All rights of reproduction in any form reserved. ISSN 0026-2862 Printed

in Great

Britain

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PITTMAN AND DULING

hemoglobin for oxygen (O,), shifting the oxyhemoglobin (HbOJ saturation curve to the right. Both the rightward shift in the saturation curve and the vasodilation tend to raise tissue oxygen tension (PO,). The vasomotor effect of CO, has been explored by a number of investigators both in whole animal experiments (Daugherty et al., 1967; Kontos et al., 1968; Kontos et al., 1970; Kontos et al., 1971) and in microcirculatory preparations (Duling, 1973a; Kontos et al., 1971; Krogh, 1920; Raper et al., 1971), but there are only two reports in the literature on quantitative measurementsin the microcirculation of the influence of CO, on tissue oxygen levels (Duling and Berne, 1970; Duling, 1973a). By adding CO2 to the solution suffusing the hamster cheek pouch, Duling (1973a) demonstrated that the vascular smooth muscle cells relaxed but continued to respond to changesin 0, demand in the appropriate fashion, although at higher PO, values than are usual. Although the percentage Hb02 ( %HbOz) was not measured, these results could be explained both by rightward shifts in the HbO, saturation curve induced by CO, and by the vasodilatory effectsof COZ. In order for there to be an effectof CO2 on the oxygen saturation curve of precapillary blood, CO, must diffuse acrossvascular walls separating suffusion solution from blood. Becauseof its high solubility it seemsreasonablethat the flux of CO, across thin-walled vessels might be relatively large. Changes in suffusion solution PCO, give rise to predictable alterations in intraerythrocytic pH, so that shifts in the blood oxygen saturation curve are due to changesin pH as well as X0,. The present study was undertaken to determine the contribution of changesin the affinity of hemoglobin for 0, to the overall effect of CO, on tissue oxygen delivery. These measurementswere made possible by the development of a new video densitometric technique (Pittman and Duling, 1975a,b) that allows the direct measurementof intravascular hemoglobin oxygenation in individual blood vesselsin the microcirculation. Sincechangesin CO, were brought about in the suffusion solution, we studied the equivalent of tissue PC02 alterations, whereas others have studied changesin arterial blood CO, levels. METHODS In this investigation 18 male golden hamsters weighing between 85 and 130 g were used. They were initially anesthetized with sodium pentobarbital (60 mg/kg, ip), supplemented during surgery via injection into a cannulated femoral vein. The trachea was cannulated, and the hamsters breathed spontaneously. Preparation of the cheek pouch was carried out as described previously (Duling, 1973a).The pouch was everted, spread,pinned on a Lucite pedestal,and slit longitudinally for viewing as a single layer. The surface was suffused with a warm, bicarbonatebuffered physiological salt solution with a millimolar composition of NaCl 131.9; KCl, 4.7; CaCl,, 2.0; MgSO,, 1.2; and NaHCO,, 18.0.The solution had beenpreviously equilibrated with a gas mixture containing 95 % nitrogen and 5 % CO*, and pH was adjusted to 7.35 at 37”. Temperature was measured on the lower surface of the pouch with a Yellow Springs telethermometer and thermistor, and was maintained at 37” by heating the incoming fluid. The hamster’s rectal temperature was maintained at 37” with a heat exchanger coil under the animal and with incandescentheating.

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213

Suffusion solutions were equilibrated with gasesof various composition to give the desired PO2 and PCOz in the supply reservoir. Differences betweenthe gascomposition of the solution over the pouch and the reservoir were minimized by a rapid circulation of fluid over the pouch surface. Solution reservoirs were equilibrated with a gas mixture nominally containing either 1,5, or 10% COz and the balance nitrogen. The resulting solutionP0, ranged from 5-l 5 mm Hg asdetermined by measurementwith the 0, microcathode. PCOz determinations were made on capillary tube samplesobtained from the solution flowing over the tissue surface. The determinations were made on a Radiometer Model PHA 927B gasmonitor with a X0, macroelectrode. The mean + SEMs of X0, for the three CO, mixtures usedabove were 10 f 2 (n = 5), 32 a 2 (n ): lo), and 75 t 3 (n = 5) mm Hg, respectively. These are comparable to the expected values of 7.0 f 0.1, 35.1 + 0.1, and 70.3 f 0.1 mm Hg basedon the observedbarometric pressureand the fractional CO, composition. In the solutions in which 1% and 10% CO, were used, the sodium bicarbonate concentration was adjusted to give a pH of 7.35 and the appropriate amount of sodium chloride was added or deleted to avoid changesin osmolarity. Measurement of Oxygen Tension PO, measurements were made using oxygen electrodes of the type developed by Whalen et al. (1967). Electrodes had tip diameters ranging from 2 to 5 pm and were polarized with 0.7 V dc. Currents, measuredwith a Keithley 602 picoammeter, ranged between IO-” and IO-lo A during equilibration of the solution with a gas containing 21% Oz. Electrodeswerecalibrated before and after measurementsin suffusion solutions equilibrated with gasesof known composition. Perivascular PO, measurementswere made on the external walls of the microvessels as previously described with the electrodes placed on the vessel wall at the midpoint in the vertical plane with the tip indenting the surface (Duling and Berne, 1970; Duling, 1972). Duling and Berne (1970) have previously shown that a rather small transmural gradient in PO, (l-2 mm Hg) exists in arterioles of this preparation, so that perivascular PO, is a good index of intravascular POz. Measurement of Fractional Oxyhemoglobin Fractional HbO, in microvesselswas determined by a video densitometric technique reported earlier (Pittman and Duling, 1975b).The microcirculation was observed with a Leitz Labolux II microscope, using long working distance objectives between 3.5 and 50x. The microscope image wasviewed with a Cohu closedcircuit television system, video signals corresponding to light intensity were processed by a video analyzer (Colorado Video, Inc., Model 321), and the dc output from this device was registered on a chart recorder (Brush Model 260). The microcirculatory sceneswere viewed on a Conrac TV monitor. The densitometric technique consisted of measuring the amount of light transmitted through a single microvessel (I) relative to the adjacent tissue (lo). At each measuring site, sequential determinations of optical density [D = log(Z,/Z)J were made at three wavelengths: 555, 546, and 520 nm. The amount of light transmitted through a vessel at the first wavelength is dependent on the relative amount of oxy-and &oxyhemoglobin present in the blood. Light transmission at the latter two isobestic wavelengths is

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independent of oxygen saturation of the hemoglobin and with appropriate manipulation yields a correction factor (B), related to the amount of light scatteredby the red cells and other nonspecific light losses (Pittman and Duling, 1975a). Fractional or percentage oxygen saturation of the hemoglobin is then linearly related to the corrected optical density ratio: S = rn(Dss5 - B)/(D546 - B) + b, where m and b are constants dependent on the wavelengths and optical system used. The method has been shown to yield an accurate (+4%) estimate of percentage saturation and is not influenced by vesselsize, hematocrit, or blood flow. Currently, only steady state determinations of % HbOz can be made, and reliable estimates of intravascular HbOz during transient situations or in caseswhere spontaneous vasomotion exists at the measurement site are not possible. To minimize transients related to vasomotion, only dilated vesselswere studied. Measurement of Diameter and Velocity

Diameter measurementsof microvesselswere made with a Vickers image-shearing eyepiececoupled to a IO-turn potentiometer. The voltage drop on the potentiometer was recorded on a Brush 260 recorder. The reproducibility of this system was +I pm using a 50x objective lens, with which most measurementswere made. Red cell velocity was measured with a rotating prism image-streaking eyepiece (Monroe, 1966). This device was calibrated by passing objects across the microscope field at known rates, and the estimated uncertainty in measurementsis within +5 % of the observed velocity. Hill Plots of Saturation Curves

Empirically, the two-parameter Hill equation, S/(1 - S) = K(PO$, where S is fractional oxygen saturation, provides a convenient description of the saturation curve Jn the range S = 0.1 to 0.9 (Antonini and Brunori, 1971). Taking logarithms of both sides of this equation yields a linear relation between log [S/(1 - S)] and log POz, where the slope, n, is called the Hill coefficient and the intercept on the ordinate is 1ogK. P,,, the PO2 at which the saturation is 50% is inversely related to the affinity of Hb for O2 and is given by log P,,, = -(logK)/n. Data pairs of fractional HbOl and PO, from each seriesof experiments were plotted as apparent HbO, saturation curves using Hill coordinates, where log[S/(l - S)] is the ordinate and logP0, the abscissa. ‘The Hill pIots presentedin this report were anaIyzed by standard linear regressiontechniques (Zar, 1974). The expected uncertainty for determining PsOby the combined video densitometric and PO, microelectrode measurementscan be estjmated by considering the respective uncertainties in the two measurements(+_4y0 HbOz and +2 mm Hg POJ in relation to the Hb02 saturation curve. Most measurementswere made on the steepportion of the saturation curve and here we estimate the instrumental uncertainty in P,, to be k2.5 mm Hg. Experimental Protocol

After surgery, the hamster was placed on the microscope stageand allowed to stabilize for 1 hr with the tissue exposedto a suffusion solution PO, of 5-l 5 mm Hg. At the end of the equilibration period, a segmentof the microcirculation was chosen which

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215

contained arterioles of from 20-60 pm inside diameter and which could be followed for 5-10 mm downstream of someinitial observation site. This particular sizerange was chosen because measurement of intravascular hemoglobin oxygenation is presently limited to vesselsof from 20-100~pm inside diameter (Pittman and Duling, 1975b). Subsequent vasodilation with adenosine resulted in diameters of the largest vessels (ca 60 pm) near the lOO+m upper limit. For each experimental condition imposed, measurements of perivascular POZ, intravascular y0 HbO,, inside diameter, and red cell velocity were made at each of three or four sites along the length of a vesseland its branches. All measurementswere made under steady state conditions in which the above four parameters remained constant during measurement.PO, and % HbO, were measured simultaneously. Each animal was subjected to two experimental situations following the initial stabilization period. The above measurementswere performed at the selectedsites under the first condition and then a change was made in the gas composition of the suffusion solution flowing over the cheek pouch. Following a 30-60-min adjustment period, comparable measurements were performed on the samesitesas before under the new conditions. The gaseous composition of the suffusion solution was changed between 1,5, and 10% CO;?with the balance nitrogen, and in most casesit contained 10e4-M adenosine. This is a supramaximal dose and eliminates vasomotor effectsof the carbon dioxide. Drugs used were: acetylcholine chloride (Sigma), adenosine (Sigma), elodoisin (CIBA), and papaverine hydrochloride (Sigma). RESULTS Longitudinal Gradient in PO, and % HbO,

As stated in the protocol, measurementsof PO, and % HbO, were made on the arterial side of the circulation at progressively more downstream locations. In all experiments PO2 and % Hb02 decreasedas more distal siteswere measured,and the % HbOz decreasedin a manner predictable from the measuredPO2 and the HbO, saturation curve for hamster blood. These observations were found at all levels of PCO, used. An example of the longitudinal gradient in % HbOz and PO, is illustrated in Fig. 1 which shows % HbO,-PO, data pairs from an experiment in which suffusion solution PCO, was increased from 32 (open triangles) to 75 mm Hg (solid triangIes). The two theoretical 0, saturation curves correspond to the adjacent PC02 values in mm Hg. For eachPC02, the points labeled L, S,and T correspond to simultaneous measurements of % HbOz and POZ at large, small, and terminal arteriolar sites, respectively. These data represent the progressive release of oxygen (fall in % HbOz and P0.J from precapillary blood as it courses through arteriolar vessels.That is, HbOz was desaturated by combined 0, loss to parenchymal cell metabolism and to the suffusion solution. The altered saturation curves observed when suffusion solution PCO, was increasedmust be in responseto the relatively rapid precapillary COZexchangebetween arteriolar blood and suffusion solution. All the % Hb02-PO2 data pairs collected in this study are plotted on Hill coordinates in Fig. 2. The straight lines in eachpanel of this figure are least squaresregression lines. These Hill plots of apparent HbOz saturation curves are graphic evidence of the axial

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po2

(mm

Hg)

FIG. 1. 0, saturation curves for an experiment in which suffusion solution PCOL was maintained first at 32 mm Hg and then at 75 mm Hg in the presence of 10-9 M adenosine. Points correspond to simultaneous measurements of percentage HbOz and POZ at the same progressively downstream arteriolar sites (L = large arteriole, S = small arteriole, and T = terminal arteriole). Solid curves are theoretical saturation curves predicted from measured suffusion solution PCO, and Severinghaus blood gas calculator. Decreases in saturation and PO2 reflect precapillary O2 lossesto suffusion solution and tissue metabolism. TABLE I LONGITUDINALGRADIENTIN

% co2 5 5‘+ 5 1 5 10

Large Arterioleb PO2(mm I-W 51.9 f 3.6 43.8 + 2.9 55.2 + 4.3 48.3 + 5.3 57.0 + 5.0 62.6 f 4.3

PO*AND %HbO,”

APO,” (mm W 7.7 f 2.2 8.1 + 1.9 12.9 + 1.4 15.8 + 2.1 14.2 + 3.6 6.9 + 1.6

Large Arterioleb % HbOz

A %HbOz”

81.7 * 4.9 81.2 + 3.6 81.4 + 5.9 78.2 f 5.3 82.8 f 5.0 79.7 +_2.0

21.4 + 6.3 25.7 -+-5.3 21.2 t_ 3.5 20.0 * 3.5 14.6 +_1.8 13.6 + 2.6

LIData are expressed as mean Jr standard error. bPOZ and HbOz for large arterioles are mean values at upstream measure merit sites for each series of experiments. c APO2 and AHbO, are means of differences inPOz and HbO, at the upstream (large arteriole) and downstream (small arteriole) sites for each series of experiments. Direction of gradient is the same for all entries: POZ and HbOz are less at downstream sites. All APO,‘s and AHbO,‘s are significantly different from zero by t test (P = 0.05). d No adenosine in suffusion solution; all other entires in presence of 10d4-M adenosine.

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SATURATION

decreasein precapillary intravascular oxygen levels, since, in general, the higher values of PO, and ‘A HbOz were measured at more proximal sites in the arteriolar network. Figure 2 will be described in more detail later. Thesedata are also presentedin Table 1 as paired differences of large arteriole minus small arteriole POZ and y0 HbOz, where the large arteriole was the upstream (proximal) measurementsite and the small arteriole the downstream (distal) site. The control values for upstream PO, (mean = 53.1 _+3.0 mm Hg) and % HbO, (mea.n= 80.0 & 0.7 %) were consistent for all six experimental 10

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FIG. 2. Plots of apparent Hb02 saturation curves on Hill coordinates (Hill plots) for simultaneous measurements of arteriolar percentage HbOz (video microdensitometer) and POz (0, microcathode). A. Circles (and line labeled 5) refer to suffusion solution aerated with 5 % CO2 and containing 1O-4M adenosine. Squares (and line labeled 5*) refer to 5 % CO, and no adenosine in suffusion solution. B. Five per cent CO, and 10m4Madenosine in suffusion solution (circles and line 5); 1% CO, and 1O-4M adenosine (triangles and line 1). C. Five percent COz and lo-“ Madenosine (circles and line 5); 10% CO, and 30d4Madenosine (diamonds and line 10). Slopes and intercepts for least squares regression lines are given in Table 2.

conditions. In addition, all the paired differences of both PO, and % HbO, entered in Table 1 were positive, indicating a flux of oxygen leaving blood, and these differences were all significantly different from zero by Student’s t test (P = 0.05). Efect of Sustained Vasodilation

In order to dissociate the effect of changesin PCOz on the HbOz dissociation curve from their vasomotor effects.on vascular diameter, experiments in which PCOz was altered were conducted under conditions of sustained vasodilation. The vasodilatory properties of several agents were tested (acetylcholine, adenosine, elodoisin, and papaverine) and only 10M4-Madenosine was capable of producing a sustained (>I hr)

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PITTMAN AND DULlNG

vasodilation when administered continuously in the cheek pouch suffusion solution. Higher concentrations of adenosine did not elicit further dilation. The effectsof adenosineon the HbO, dissociation curve were tested as shown in Fig. 2A. In the first seriesof experiments, the suffusion solution was gassedwith 5 % COZ95 % Nz and there was no adenosine present. The protocol given above was followed in which PO, and % HbO, were measured simultaneously. Figure 2A (solid squares) shows a Hill plot which to good approximation is a linearization of the HbO, dissociation curve. Values for the slope of the linear regression lines (n) and Pso are given in Table 2 (mean + SEM). The sameprotocol was followed with 10e4M adenosine in the TABLE 2 HbOz SATURATIONCURVEPARAMETERSFROMHILLPLOTS

% co2

5 5” 5 1 5 10

Pso + SEM (mm Hd

28.4 + 29.1 + 31.1 + 25.3 + 28.8 + 40.9 *

3.2 (19) 1.7 (15) 2.6 (13) 2.8 (13) 2.9 (12) 3.9 (14)

Predicted P5d (mm W

26.6 26.6 26.6 15.2 26.6 40.0

nb

rc

1.9 + 0.7 2.7 rt 0.6 2.4 + 0.4 2.0 + 0.5 2.3 + 0.5 2.4 + 0.7

0.54 0.75 0.85 0.76 0.79 0.69

y Pso was predicted from the data of Hall (1966), Ulrich et al. (1963), and Volkert and Musacchia (1970). COz-induced shifts inPsOwereestimated from the data of Severinghaus (1966) for human blood. * n, the Hill coefficient, has a predicted value in the range 2.7-3.0 (Tyuma et al., 1972). e r is least squares correlation coefficient, All r values are significantly different from zero (P = 0.01) except for the first entry (P = 0.02). Numbers in parentheses in second column are number of observations used for statistical computations. d No adenosine in suffusion solution; all other entries in presence of lO-4-M adenosine.

suffusion solution. There wasusually a brief period (ca. 15 min) during which arteriolar vasomotion occurred after introduction of the adenosine. After stabilization, similar measurementswere performed on the samesites. The PO, and ‘A HbO, are plotted in Fig. 2A (solid circles) according to Hill’s equation. The P5,, in the control experiments (29.1 + 1.7 mm Hg) was not significantly different from that with adenosine suffusion (28.4 +_3.2 mm Hg) at the P = 0.05 level. The Hill coefficient (n) changed from 2.7 + 0.6 to 1.9 f 0.7. Ordinarily this large change in n would indicate a corresponding alteration in the affinity of Hb for 0,. However, the large standard errors on n in this casedo not allow oneto makea definitive statement on this point. The two other entries of comparable data in Table 2 for 5 % CO, and 10m4M adenosine in the suffusion solution suggest values of n (2.4 + 0.4 and 2.3 + 0.5) in better agreement with expectedvalues (2.7-3.0) and no change in P,. can be attributed

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to adenosine.Thus, we found no evidencefor a direct effectof adenosineon the measured saturation curve. Decreases in PCO,: 5 to 1% CO?

Experiments were performed in which the composition of the gas aerating the suffusion solution was decreasedfrom 5 to 1% COZ,both in the presenceof 10M4Madenosine. Hill plots of these data are shown in Fig. 2B. Solid circles correspond to 5 % CO, in the suffusion solution and solid triangles to I % CO,. The 5 % CO2 data were used as a referenceor control situation inlater data analysis. No changesinvascular diameter were noted on changing from 5 to 1 % CO, in the suffusion solution and none were expected since the cheek pouch vasculature was dilated to the sameextent by 10P4-Madenosine during both suffusion periods. Mean P5,, decreasedfrom 31.1 + 2.6 mm Hg in experiments using 5% CO* to 25.3 f 2.8 mm Hg in those using 1% CO,. The former P,O was not significantly different (P = 0.05) from the predicted value of 26.6 mm Hg, but the latter value was significantly higher (P = 0.05) than the expected value of 15.2 mm Hg. The predicted difference in P,, on going from 5 to 1 y0COZsuffusion, assuming full CO, equilibration between blood and suffusion solution, is 1I .4 mm Hg, while we only observed a 5.8 mm Hg decreasewhich was not statistically significant at the 5 % level. Since the estimated uncertainty in our P5,, determination is +2.5 mm Hg, it was anticipated that we would easily be able to observe the 11.4 mm Hg predicted PsO difference. Values for the Hill coefficient did not differ significantly (P = 0.05) from each other or from the expected range of values [Table 2). increases in PCOz: .5 to 10 % CO,

A third group of experiments was performed in which the composition of the gas aerating the suffusion solution was elevated from 5 to 10‘A C02, in the presenceof 10e4 A4 adenosine. The results of these experiments are plotted according to the linearized form of Hill’s equation in Fig. 2C. Linear regression lines are given for the data with solid circles corresponding to 5 % CO, and diamonds to 10% CO,. Again, no changes in vascular diameter were noted when the gas was changed from 5 to 10% CO2 since the preparation wasvasodilated throughout eachexperiment. PsOincreasedfrom 28.8 + 2.9 mm Hg in experiments using 5 % CO, to 40.9 + 3.8 mm Hg in those using 10% CO,. The latter PsOwas statistically greater than the PsOwith 5% CO, at the 5% level by a paired t test. The difference in PsOpredicted from the saturation curves was 13.4 mm Hg compared to an observed difference of 12.1 mm Hg, an agreement which is consistent with the estimated instrumental uncertainty in P,,, determination. Values for the Hill coefficient did not differ significantly (P = 0.05) from each other or from the expected range of 12(Table 2). DISCUSSION The data presented above are the first direct measurementsof CO,-induced shifts in the HbO, dissociation curve at the level of microcirculation. As expected,changesin the PCO, of the suffusion solution flowing over the cheek pouch brought about predictable alterations in the affinity of Hb for 02, although the PsOestimated for 1% CO, in the suffusion solution was considerably greater than expected. A recent study (Case

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et al., 1975) dealing with the effect of altered PCO, on coronary sinus POz and 0, saturation for dog heart yielded results consistent with those reported here. These authors demonstrated that profound changesoccur in the 0, content of coronary sinus blood as a result of changesin arterial PCO,. A longitudinal gradient in both POz and % HbO, was observed in all animals. That is, precapillary lossesin 0, existed for different levels of CO2 in the suffusion solution and in the presenceand absenceof adenosinein the solution. This observation confirms earlier reports of this phenomenon (Duling and Berne, 1970; Duling, 1972; Duling, 1973a)and in addition demonstratesthat the longitudinal gradient in PO, is associated in a predictable way with concomitant decreasesin % HbO,. Going from large to small arterioles, averageoxygen saturation decreasedfrom 82 to 61 y0 in the 5 ‘A COz experiments, 78 to 58% in 1% CO,, and 80 to 66 % in 10% CO,. The dependenceof the longitudinal gradient on velocity is indicated by the fact that in one experiment of this series,the blood flow in the cheek pouch spontaneously increasedfrom one steady level to another, a commonly observed phenomenon in this preparation. The ratio of control and experimental velocities was 1.5 and the corresponding (inverse) ratio for the decreasein % HbOz over this segmentwas 1.4. The close agreement indicates that a fairly simple model (Middleman, 1972) is not unrealistic in describing precapillary 0, exchange.Quantitation of this nature needsto be pursued more systematically now that direct measurements of the appropriate convectiondiffusion parameters can be made at the level of individual microvessels. Although blood vesseldiameter and red cell velocity were routinely measuredin this study, thesedata were usedprimarily asan index of preparation stability and normality. Inside diameters increased by approximately 32 % for large arterioles and 6 1% for small arterioles over control values in responseto 10F4M adenosine. There were concomitant increasesin red cell velocity which appearedto dependmarkedly on the overall perfusion of the cheek pouch. Qualitatively, red cell velocities were uniformly distributed between 1 and 12 mm/set and were noticeably lower for the smaller vesselsin any given preparation. No attempt has been made to usethese data in a quantitative model analysis of peripheral O2 exchange. The first group of control or baselineexperimentswasconducted to determine whether the adenosine levels used to dilate the cheek pouch microvasculature influenced the HbO, dissociation curve. Figure 2A and Table 2 show that both P,, and it were not significantly different with 5 y0 CO, in the suffusion solution in the presenceor absenceof 1O-4M adenosine.We conclude that this concentration of adenosinehas no measurable effect on the affinity of hemoglobin for 0,. It has been shown by others (Rubinstein et al., 1956; Duhm, 1972; Duhm, 1974) that incubation of red cells in adenosine and inosine at concentrations about 100 times that used by us leads to significant increases in intracellular 2,3-diphosphoglycerate and hydrogen ions. These increases tend to smft the HbOz dissociation curve to the right and thus decreasethe affinity of hemoglobin for Oz. Basedon the work of Duhm (1972) we have estimated that the influence of low4.J4adenosineon Psoshould be very small (~1 mm Hg shift), hence,physiological levels of adenosine (E 10V6M) will have negligible effects on red cell oxygenation. The P,, for 10% COz in the suffusion solution is significantly greater (P < 0.05) than that for the 5% CO, control value and it is not different, within experimental uncertainty, from the predicted value based on measured solution PCO,. This result

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suggeststhat no significant disequilibrium exists between blood and solution PCOz at the measurement sites in the cheek pouch. It might be anticipated that the rapidity of CO, entry and reaction within the red cell would lead to such a situation (Forster and Steen, 1968). Although P,,, for 1% CO, in the suffusion solution is less than that for the 5 % COz control value, it is not significantly lower by Student’s t test (P = 0.05). Also, Pso for 1% CO, is significantly higher than would be predicted on the basis of the 10 mm Hg PC02 in the suffusion solution (column 2, Table 2). This suggeststhat the blood at this point in the microcirculation may not be fully equilibrated with the COz in the suffusion solution. This possible disequilibrium in CO, between blood and suffusion fluid may not be unreasonable since Forster and Steen (1968) have reported that the half-time for the Bohr shift causedby a decreasein PCO, (0.35 set) is about three times that caused by an increasein PCO,. Most observations on microvesselswere made about 1 cm from the arterial input to the cheek pouch. Assuming an average red cell velocity of about 10 mm/set, this would mean that the blood had been exposedto the PCO, of the suffusion solution for about 1 set at this point. It is known that oxygenating blood facilitates the egressof COz, (Hill et al., 1973)but in our caseit is not clear to what extent the release of CO, by blood is retarded by a concomitant decreasein PO2 and % HbO,. One further difference between the experiments using 10 and 1% CO, in the suffusion solution is that the diffusion gradient for CO, between solution and blood for the 10% CO, solution is about three times that for the 1% COz solution. Also, since CO, is being continuously produced by parenchymal cell metabolism, it is probably more difficult to obtain equilibration between suffusion solution and blood when the PCO, of the suffusion is lowered than when it is raised. Direct measurementsof COz exchange at the microcirculatory level may ultimately provide important information on the transport of this respiratory gas between tissue and blood. However, at this time no techniques are available to explore the dynamics of CO, exchange in single microvessels. A careful consideration of our findings for 02, however, can be of help in estimating the behavior of COz. If one knows temperature, pH, PCOz and 2,3-DPG for a volume of blood, then one can define with reasonable precision the HbO, saturation curve. In our experiments we have controlled temperature, assumednegligibly small changesin plasma pH and red cell 2,3-DPG, and measuredPO, and % HbO,. Therefore shifts in the HbO, saturation curve may be assumedto be due principally to changesin PCO, with, of course, concomitant changesin RBC pH. One can imagine a family of saturation curves, each one valid for a given PC02, with temperature and 2,3-DPG fixed at appropriate values. A % Hb02-PO2 data pair can be placed on only one of these saturation curves and, by determining the PCOz corresponding to that curve, an estimate of blood PCO, can be obtained. For each point in Fig. 2, PCOz was estimated using the Severinghaus blood gas calculator (radiometer) assuming a plasma bicarbonate concentration of 21 mEq/L. The Pso for hamster blood (27.8 mm Hg) at pH 7.4 and T = 37” was taken as the averageof values from Hall (1966), Ulrich et a/. (1963), and Volkert and Musacchia (1970). Estimates of PCOz for large and small arterioles were obtained in this way for suffusion with solutions equilibrated with 1, 5, and 10% CO,. Comparison of large and small arteriolar PCOz values yielded demonstrable differences in PCO, in the expecteddirection, and thus suggesteda longitudinal gradient of CO, in theseprecapillary

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vessels.However, only the large to small arteriolar difference in PCO, for 10% CO, suffusion, -14 f 5 mm Hg (mean + SE), that is an uptake of CO, by blood, was sign& ficant at the P = 0.05 level. Further experiments designed to measure intravascular pH or PCO, directly will be required to confirm this finding. The existenceof a longitudinal gradient in PCO, may have an important implication with regard to regulation of cerebral blood flow. It is currently held that COZ plays a major role in determining arteriolar tone in the cerebral circulation (Severinghaus et al., 1971/72)through its relation to H+ ion. Using a convection-diffusion model for CO, similar to that presented by Duling and Berne (1970) for oxygen, the longitudinal gradient in PCO, will be directly proportional to the ratio of metabolic rate to blood velocity and we can hypothesize the following mechanism. As blood passesthrough the arterioles, the increase in PC02 at any point is a function of the combined effects of blood velocity and the diffusion gradient for CO, from the tissuesto blood, A reduction in velocity would raise intravascular PCO, and thus the PCO, of the vascular smooth muscle.Arteriolar smooth muscleis sensitiveto increasedPC02 at levelsnormally found in the microcirculation (Kontos et al., 1971/72),so that the increasedPCOz would produce dilation of the arterioles and an increasein the velocity of flow. A new steady state would then be achievedat a larger arteriolar diameter. Similarly, an increasein metabolic rate of tissue would produce an increase in the carbon dioxide diffusion gradient, more rapid gain of carbon dioxide by the precapillary blood and a relaxation of vascular smooth muscle. An indication that the velocity dependencein this scheme is approximately correct is obtained by considering the example earlier in the discussion in which blood velocity spontaneously increased.The ratio of control to experimental velocities was 1.5 and the corresponding (inverse) ratio for the changein predicted PCO, over this segmentwas 3.3, a result which is qualitatively in the proper direction. It is perhaps instructive to consider a typical red cell entering the cheek pouch and to follow the exchangeof O2 and COZ between it and the adjacent tissue and suffusion solution. Although we did not measure arterial blood gasesin this study, Volkert and Musacchia (1970) reported mean arterial values in anesthetized hamsters of PO, = 71 mm Hg and PCO, = 56 mm Hg, and Duling and Berne (1970) have reported a mean arterial POz of 69 mm Hg. The mean large arteriolar PO, found in the present study was 53 + 3 mm Hg and indicates some upstream O2 losses to tissue metabolism and the suffusion solution 0, sink (PO, = 5-15 mm Hg). Thus, the walls of larger arterial vesselsupstream of our measurement sites possesseda finite permeability to 0, and probably also to CO, since it is much more soluble than 0,. The decreasein arteriolar PO, and % HbOz shown in Fig. 1 and Table 1 is simply a reflection of 0, lossesproviding for local tissue metabolism and O2 moving into the suffusion solution. With regard to CO, exchange, the typical red cell, upon entering the cheek pouch at an arterial PCOz of about 56 mm Hg, was exposed to a suffusion solution PCOz of 10, 32, or 75 mm Hg. Thus, the initial diffusion gradients for CO, were 46, 24, or -19 mm Hg, respectively, where the positive values correspond to CO, movement out of blood. Large arteriolar PCO, was estimated by the method previously described, and values of 31, 43, and 76 mm Hg were obtained for suffusion solution PC02’s of 10, 32, and 75 mm Hg, respectively. Thus, our estimatesshow about 54 % equilibration between blood and suffusion solution PCOz for the two lowest PCOz solutions and essentially complete equilibration for the highest PCO,. The fairly large disequilibrium

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for the 1% COz solution is consistent with the large deviation of observed Pso from the predicted value (Table 2) and with the observation that all Pso values for 5 % CO2 suffusion (Table 2) are higher than predicted by about 3 mm Hg. In summary, we have confirmed the existenceof a longitudinal decreasein arteriolar PO, and have shown that it is related in a predictable way to an accompanying decrease in hemoglobin 0, saturation. Shifts in the 0, saturation curve were produced by altering the local CO, environment in a manner analogous to tissue CO, changes. Evidence was also presented which suggeststhat significant CO, exchange can occur across the walls of precapillary vesselsand that a longitudinal gradient in CO, might exist which could have important implications with respectto local regulation of blood flow.

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