ATP overflow in skeletal muscle 1A arterioles

Abstract

The purpose of this study was to investigate the sources of ATP in the 1A arteriole, and to investigate age-related changes in ATP overflow. Arterioles (1A) from the red portion of the gastrocnemius muscle were isolated, cannulated and pressurized in a microvessel chamber with field stimulation electrodes. ATP overflow was determined using probes specific for ATP and null probes that were constructed similar to the ATP probes, but did not contain the enzyme coating. ATP concentrations were determined using a normal curve (0.78 to 25 micromol l(-1) ATP). ATP overflow occurred in two phases. Phase one began in the first 20 s following stimulation and phase two started 35 s after field stimulation. Tetrodotoxin, a potent neurotoxin that blocks action potential generation in nerves, abolished both phases of ATP overflow. alpha1-Receptor blockade resulted in a small decrease in ATP overflow in phase two, but endothelial removal resulted in an increase in ATP overflow. ATP overflow was lowest in 6-month-old rats and highest in 12- and 2-month-old rats (P<0.05). ATP overflow measured via biosensors was of neural origin with a small contribution from the vascular smooth muscle. The endothelium seems to play an important role in attenuating ATP overflow in 1A arterioles.

Figure 1. The vessel chamber

Diagram of the vessel chamber showing the cannulated 1A arteriole and the positioning of the ATP probe, the null probe and the stimulating electrodes.

Figure 2. Representative data from one arteriole show the vessel diameter changes with 60 Hz, 200 impulses, 32 mA field stimulation repeated five times

The vessel diameter changes were consistent and measurable during field stimulation.

Figure 3. Summary of data show the lifetime of the ATP probe over repeated usage

Probe electrical current (pA) response to increasing doses of ATP on the first day of use to the third day of use (A). Probe responsiveness was well-maintained over 2 days of use, but began to lose sensitivity at the lower concentrations of ATP by day 3 (P > 0.05). Overall responsiveness (B) of the ATP probe declined by the third day of use.

Figure 4. Sum of the integrals over 60 s following field stimulation at different amperages and frequencies of field stimulation

A, sum of the integrals for 60 Hz, 200 impulses at 9 mA to 32 mA constant current field stimulation. Small amounts of ATP were detected at 9 mA to 30 mA, but the best amperage was 32 mA for detection of ATP. B, the sum of the integrals for different frequencies (200 impulses, 32 mA). The best frequency for detection of ATP was 60 Hz.

Figure 5. Representative raw data tracing of the ATP and null probe response to 60 Hz field stimulation and 60 s following the stimulation period

A, active field stimulation resulted in electronic interference with the probes, but immediately following the end of stimulation, there was an increase in the electrical potential detected by the ATP probe. This increase was shortly followed by an increase in the null probe which rapidly exceeded the electrical potential detected by the ATP probe. This was reversed to a small degree in the last 10 s of recording. B, a summary of ATP overflow following field stimulation in gastrocnemius 1A arterioles from 6-month-old male rats (n = 6).

Figure 6. Summary of the results from adding tetrodotoxin to determine the neural component of ATP overflow (n = 2, 6-month-old rats)

ATP overflow increased briefly following field stimulation with no blocking (A). After the addition of the vehicle (citrate buffer) for tetrodotoxin, ATP overflow increased significantly between 10 and 45 s following field stimulation (B). After the tetrodotoxin was added, no ATP overflow was detected following field stimulation (C).

Figure 7. Summary of the ATP overflow to field stimulation in endothelium-intact (n = 6) and endothelium-removed (n = 3) arterioles

A, ATP overflow for 60 s following field stimulation in each arteriole. The endothelium-removed arterioles are the dashed lines and the endothelium intact arterioles are the continuous lines. B, when the data above were represented as a mean of the sum of the integrals for the 60 s recording period, there was a significant increase in ATP overflow when the endothelium was removed (P < 0.05; * denotes difference from endothelium-intact arterioles).

Figure 8. Representative raw data tracing of the ATP and null probe response to the α₁-agonist, phenylephrine, and increasing doses of the α₁-antagonist, prazosin

Phenylephrine caused a large increase in the pA detected by the ATP probe. This response was attenuated by the addition of prazosin at 1.4 μmol l⁻¹ and somewhat attenuated by 14.29 μmol l⁻¹, but high doses of prazosin (142.9 μmol l⁻¹) also attenuated the response of the null probe. Therefore, in subsequent experiments, the dose of prazosin used was 1.4 μmol l⁻¹.

Figure 9. Summary of the results from removing the endothelium and blocking with the α₁-antagonist, prazosin (n = 4)

A shows the change in ATP overflow with prazosin from the control condition. Prazosin attenuated ATP overflow in the late phase in 2 out of 3 arterioles studied (P > 0.05). B shows the mean response for the control and prazosin condition. Blocking α₁-receptors with prazosin resulted in a small attenuation of ATP overflow.

Figure 10. Mean ATP overflow from 1A arterioles from young adult, mature adult and middle aged rats

Arterioles from young adult (2 months old, n = 7) animals (A) had high ATP release in the first 20 s following field stimulation and again at the 25 to 60 s time point. Mature adult rats (6 months old, n = 4; B) had a small ATP overflow from 0 to 10 s and again starting at 35 s following field stimulation (P < 0.05 different from 2-month- and 12-month-old groups). ATP overflow in middle aged animals (12 months old, n = 5; C) followed a similar pattern as occurred in the younger animals, but the magnitude of the response was approximately 4-fold higher (P < 0.05), compared to 6-month-old animals. *P < 0.05 different from 2-month- and 12-month-old animals.