Cell-specific differential modulation of human trabecular meshwork cells by selective adenosine receptor agonists

Abstract

Activation of A1 and A2A subtype adenosine receptors (AR) likely exert opposing effects on outflow of aqueous humor, and thereby, on intraocular pressure. Selective agonists of adenosine receptor (AR) subtypes have previously been applied to trabecular meshwork (TM) and Schlemm's canal (SC) cells to identify the site(s) of differential purinergic modulation. However, the apparent changes in volume monitored by previously measuring projected cell area might have partially reflected cell contraction and relaxation. In addition, whole-cell current responses of the TM cells previously described were highly variable following application of selective A1, A2A and A3 agonists. The complexity of the electrophysiologic responses may have reflected cell heterogeneity of the populations harvested from collagenase digestion of TM explants. We now report measurements of TM-cell volume using calcein fluorescence quenching, an approach independent of contractile state. Furthermore, we have applied selective AR agonists to a uniform population of human TM cells, the hTM5 cell line. A1, but not A2A or A3, AR agonists triggered TM-cell shrinkage. Both A1 and A2A AR agonists produced reproducible increases in TM-cell whole-cell currents of similar magnitude. The results suggest that previous measurements of explant-derived TM cells may have reflected a range of responses from phenotypically different cell populations, and that the opposing effects of A1 and A2A agonists on outflow resistance are not likely to be mediated by actions on a single population of TM cells. These opposing effects might reflect AR responses by two or more subpopulations of TM cells, by TM and SC cells or by inner-wall SC cells, alone.

Fig. 1

Cell volume (in %) normalized to value just prior to perfusion with the AR agonist. For purposes of comparison, the same set of control data are presented (open circles) in each panel. To verify the integrity and stability of the preparation, each experimental period was preceded by a 35-minute period of perfusion with iso-, hypo- and hyperosmotic solution. A, B and C: Perfusion with S-ENBA (A₁ agonist), ADAC (A₁ agonist) and Cl-IB-MECA (A₃ agonist), respectively. In this and all successive figures, uncertainties represent ±1 SE. The full names of the drugs used in the experiments of all figures are entered in Table 2.

Fig. 2

Effects of A_2A agonists on cell volume. A, B and C: Perfusion with 30 nM CGS-21680, 100 nM CGS-21680 and 50 nM DPMA, respectively.

Fig. 3

Effects of the physiologic nonselective (NS) AR agonist adenosine on cell volume. A, B and C: Perfusion with 3, 10 and 100 μM, respectively.

Fig. 4

Effects of A₁, A_2A and A₃ agonists on free intracellular Ca²⁺ concentration in A (N=11), B (N=6) and C (N=6), respectively. N is the number of coverslips studied.

Fig. 5

Effect of 100 nM S-ENBA (A₁ agonist) and 7.5 mM TEA (K⁺-channel blocker) on whole-cell currents and subsequent washout with isotonic control solution (CON). The holding potential was −40 mV, and the step pulses were to values ranging from −100 to +80 mV in 20-mV steps.

Fig. 6

Effect of 30 nM CGS-21680 (A_2A agonist) and 7.5 mM TEA (K⁺-channel blocker) after intermitting washout with isotonic control (CON) on whole-cell currents.

Fig. 7

Summary of effects (means ±SE) of AR agonists on whole-cell currents.

Fig. 8

Summary of AR effect on different cells of the eye aqueous humor outflow pathway. 100nM CGS (A_2A agonist) and 100nM Cl-IB-MECA (A₃ agonist) were used for modulation of [Ca²⁺]I, K⁺ current and cell volume. For A1 stimulation 30nM ADAC or 100nM CPA were used for [Ca²⁺]I or K+ current and cell volume, respectively. In parenthesis: 1) 100nM CPA and 2) 100nM S-ENBA were used instead; 3) unpublished data. [Ca²⁺]I was rated by changes in nM and K+ currents and cell volume by Δ % as follows: ↑ <100nM / <50%; ↑↑ =100–200nM / =50–100%; ↑↑↑ >200nM / >100%; Ø = no effect; blanks = not done.