Role of Adenosine in Cerebral Vasodilator Responses to Sciatic Nerve Stimulation

To the Editor:

We have read with interest the study by Northington and others (1992) in which they were unable to document any increase in interstitial fluid (ISF) concentration of adenosine (ADO) with sciatic nerve stimulation, nor were they able to alter CBF response to sciatic nerve stimulation with 8-(p-sulfophenyl)theophylline (8-SPT) infusion. In contrast, previous studies in our laboratory (Ko et al., 1990) have demonstrated that topical infusions of theophylline decreased the pial vascular response to sciatic nerve stimulation and that agents that increased ADO availability (e.g., dipyridamole and inosine) increased the response.

We raise the following questions regarding the accuracy of interstitial ADO levels as measured by Northington and colleagues. First, could the lack of a measurable increase in dialysis concentrations be related to lack of sensitivity of ADO measurements? The limit of detection of ADO by HPLC using ultraviolet absorbance is in the low picomolar range (Gayden et al., 1991). We calculated the amount of ADO in the perfusate sample used by Northington and associates for HPLC analysis. Our calculations, based on a baseline ADO value of 0.6 μmol/L (as reported in the study of Northington et al.), and assuming a 50% recovery, reveal that a 40 μL sample of diluted (15-fold) dialysate contained only 0.8 pmol/L, which is at the limit of detectability. Whereas a 10-fold increase was detected during global hypoxia, it would be considerably more difficult to document a small (e.g., twofold) change in ADO by the methods of Northington and others.

Second, could the lack of measurable increase in dialysis concentrations also be related to uptake and degradation of ADO? Lending credence to this possibility is the study by Van Wylen and associates (1989), which demonstrated that significant degradation of ADO occurred in the infusion dialysate. Thus, with infusion of 1 mmol/L ADO, Van Wylen and associates measured a 10-fold loss in the effluent. With infusion of 0.1 mmol/L ADO, 0.01 mmol/L ADO was recovered. Interstitial ADO concentration likewise may be lowered on the outward course of the dialysate in the dialysis tubing. Thus, this loss of ADO (either by degradation, uptake, or both) would further decrease the ability of the investigators to measure increases in ADO during sciatic nerve stimulation and impact further on the sensitivity of the HPLC analysis (vida supra).

Third, could artificially elevated resting ISF concentrations of ADO caused by tissue injury during probe insertion obscure subsequent small elevation during sciatic nerve stimulation? The investigators studied their animals approximately 60 minutes after probe insertion when ISF levels were similar to “baseline values.” However, studies by Ballarin and colleagues (1991) indicate that ISF ADO concentrations continue to decrease to considerably lower concentrations (50 to 300 nmol/L) over a 24-hour period after probe insertion. Thus, small increases in ADO concentration during sciatic nerve stimulation may have been obscured by elevated background ADO concentration caused by probe insertion.

Northington and others further noted that when they infused 8-SPT into the dialysis probe, they were unable to block the increases in CBF as measured by hydrogen clearance. The investigators found that 8-SPT caused a significant decrease in CBF during resting conditions. In contrast, Van Wylen and colleagues, when working in the same laboratory, using identical techniques, or both, noted no change in resting CBF with 8-SPT infusions (Van Wylen et al., 1988a, 1988b). Recent studies by Sciotti and associates (1992) also failed to show a change in baseline CBF with 8-SPT administration. Recently, we documented that topically applied 8-SPT attenuates arteriolar vasodilation in sensory hindlimb cortex, whereas intravenously applied 8-SPT had no effect (Meno et al., 1993). Northington and others discussed in detail the differences between topical and systemic applications of methylxanthines, but the relevant studies for comparison are those of Van Wylen and associates and Sciotti and coworkers (vida supra) who used identical methods to those of Northington and colleagues.

One argument raised by Northington and others against the role of ADO in sciatic nerve stimulation is that there is no oxygen deficit during sciatic nerve stimulation and therefore no signal for ADO release. Previous studies in cardiac tissue have shown that ADO production is linked to tissue Po₂ (Bardenheurer and Schrader, 1986; Deussen and Schrader, 1991). This relation between Po₂ and ADO production was the basis of Berne's ADO hypothesis (Berne, 1963). This issue, however, remains controversial, as recently acknowledged by Berne and colleagues (Headrick et al., 1993). For example, Gorman and colleagues (1992) have shown that there is a sustained ADO release during metabolic stimulation with norepinephrine infusion with no decrease in oxygen supply–demand ratio or with little change in cytosolic high-energy phosphate concentrations.

Northington and others used laser Doppler methods to measure CBF over a 30-second period of sciatic nerve stimulation and noted a pattern of continuously rising blood flow, which is different from the pial arteriolar response noted by us (Ko et al., 1990; Ngai et al., 1988). Northington et al. (1992) attached physiologic significance to their observations, but other explanations are possible. As outlined in our recent study (Ngai et al., 1995), we have found that laser Doppler flow follows the changes in pial arteriolar dilation. A response pattern of an initial peak followed by a decline to a steady, smaller response plateau was consistently evoked with stimulus parameters of 0.2 to 0.3 V, 5 Hz, and 0.5-millisecond pulses (Ngai et al., 1988). The response became irregular and inconsistent if stronger stimuli were used (e.g., by increasing pulse duration), partly because of the accompanying fluctuations in blood pressure, as we previously described (Ngai et al., 1988). We noted that Northington and others did not completely specify their stimulus parameters. As shown in our recent study (Ngai et al., 1995), increasing pulse duration from 0.5 to 5 milliseconds evoked a steadily rising response with no initial peak. The latter response pattern is similar to that reported by Northington and coworkers.

Lastly, Dirnagl and colleagues (1994), studying whisker barrel cortical stimulation in the rat, noted that topically applied theophylline (50 μmol/L) or adenosine deaminase reduced CBF (as measured by laser Doppler) during activation. The study of Dirnagl and others, therefore, confirms our earlier observations (Ko et al., 1990) of attenuation of the pial arteriolar response to sciatic nerve stimulation by theophylline.

In summary, we are puzzled by the results of Northington and colleagues and believe that multiple questions need to be resolved before arriving at their sweeping conclusion that ADO “does not mediate CBF changes that occur during sciatic nerve stimulation.” As Virchow observed more than a century ago, “The absence of proof does not constitute the proof of absence.”

[ Editor's note : Publication of this letter, originally received in 1994, was delayed for administrative reasons. For this, we apologize. The Editor]