Novel excitatory effects of adenosine triphosphate on contractile and pacemaker activity in rabbit urethral smooth muscle

Abstract

Purpose: Adenosine triphosphate is thought to be an important neurotransmitter in urethral smooth muscle but its physiological role is still unclear. We characterized the effects of adenosine triphosphate on contractile and pacemaker activity in rabbit urethral smooth muscle.

Materials and methods: Tension recordings were made from strips of rabbit proximal urethral smooth muscle. Membrane currents from freshly isolated smooth muscle cells and interstitial cells of Cajal were recorded using the patch clamp technique. Intracellular Ca(2+) was measured using confocal microscopy.

Results: Exogenous application of adenosine triphosphate (10 microM) evoked robust contractions that were inhibited by the type 2 purinergic receptor blocker suramin (100 microM) and the selective type 2 purinergic Y1 receptor antagonist MRS2500 (Tocris Bioscience, Ellisville, Missouri) (100 nM). Application of the type 2 purinergic Y receptor agonist 2-MeSADP (1 microM) mimicked the effects of adenosine triphosphate. When smooth muscle cells were studied under voltage clamp at -60 mV, adenosine triphosphate evoked a large single inward current (greater than 1.2 nA) but 2-MeSADP produced a small current (about 16 pA). In contrast, when interstitial cells of Cajal were held at -60 mV, they showed spontaneous transient inward currents that were increased in frequency by adenosine triphosphate and 2-MeSADP. These excitatory effects were inhibited by suramin and MRS2500. Interstitial cells of Cajal showed spontaneous Ca(2+) waves that were increased in frequency by adenosine triphosphate and 2-MeSADP. These effects were also inhibited by suramin and MRS2500.

Conclusions: Contractile effects of adenosine triphosphate in urethral smooth muscle are mediated by the activation of type 2 purinergic Y receptors on interstitial cells of Cajal.

Figure 1

Effect of suramin on EFS induced contractions of rabbit urethral smooth muscle. A, 0.3-millisecond pulse duration and 4 Hz frequency for 30 seconds (s) produced transient contraction that was attenuated by 100 μM suramin. B, contraction amplitude plotted in absence (Control) and presence of suramin in 11 experiments.

Figure 2

Effect of ATP on contractile activity in rabbit urethral smooth muscle strips. A and B, representative traces show that suramin and MRS2500 inhibited ATP induced contractions. s, seconds. C, representative record shows that 2-MeSADP mimicked ATP contractile effects. D to F, summary data.

Figure 3

Effect of ATP and 2-MeSADP on inward currents recorded in isolated urethral SMCs and ICC. A, representative trace shows that ATP but not 2-MeSADP evoked large transient inward current in urethral SMCs voltage clamped at −60 mV. s, seconds. B, mean amplitude of currents evoked by ATP and 2-MeSADP. C, representative trace shows that ATP and 2-MeSADP increased STIC frequency in ICC under voltage clamp at −60 mV. D, summarized data.

Figure 4

Effect of suramin on STICs induced by ATP and 2-MeSADP in ICC. A, representative trace shows that suramin inhibited STICs induced by ATP. s, seconds. B, mean STIC frequency induced by ATP in absence and presence of suramin. C, representative trace shows that suramin inhibited STICs induced by 2-MeSADP. D, mean STIC frequency induced by 2-MeSADP in absence and presence of suramin.

Figure 5

Effect of MRS2500 on STICs induced by ATP and 2-MeSADP in ICC. A, representative trace shows that MRS2500 inhibited STICs induced by ATP. s, seconds. B, mean STIC frequency induced by ATP in absence and presence of MRS2500. C, representative trace shows that MRS2500 inhibited STICs induced by 2-MeSADP. D, mean STIC frequency induced by 2-MeSADP in absence and presence of MRS2500.

Figure 6

Effect of suramin and MRS2500 on STICs in isolated urethral ICC. A and C, representative traces show effect at −60 mV. s, seconds. B and D, mean STIC frequency in absence and presence of suramin and MRS2500.

Figure 7

Effect of ATP and 2-MeSADP on spontaneous Ca²⁺ oscillations in isolated urethral ICC. A and B, post hoc line scan and intensity profile plots. s, seconds. C and D, summary plots on wave frequency.

Figure 8

Effect of suramin on ATP induced Ca²⁺ oscillations in isolated urethral ICC. A, post hoc line scan and intensity profile plot show effect of 100 μM suramin. s, seconds. B, mean wave frequency induced by ATP in absence and presence of suramin.

Figure 9

Effect of MRS2500 on ATP induced Ca²⁺ oscillations in isolated urethra ICC. A, post hoc line scan and intensity profile plot show effect of 100 nM MRS2500. s, seconds. B, mean wave frequency induced by ATP in absence and presence of MRS2500.

Figure 10

Representative recording shows effect of ATP on urethral contractility when applied before and during precontraction with AVP. s, seconds.