Topographical heterogeneity of K(IR) currents in pericyte-containing microvessels of the rat retina: effect of diabetes

Abstract

Although inwardly rectifying potassium (K(IR)) channels are known to have important functional roles in arteries and arterioles, knowledge of these channels in pericyte-containing microvessels is limited. A working hypothesis is that K(IR) channel activity affects the membrane potential and thereby the contractile tone of abluminal pericytes whose contractions and relaxations may regulate capillary perfusion. Because pericyte function is thought to be particularly important in the retina, we used the perforated-patch technique to monitor the ionic currents of pericytes located on microvessels freshly isolated from the rat retina. In addition, because changes in ion channel function may contribute to microvascular dysfunction in the diabetic retina, we also recorded from pericyte-containing microvessels of streptozotocin-injected rats. Using barium to identify K(IR) currents, we found that there is a topographical heterogeneity of these currents in the pericyte-containing microvasculature of the normal retina. Specifically, the K(IR) current detected at distal locations is strongly rectifying, but the proximal K(IR) current is weakly rectifying and has a smaller inward conductance. However, soon after the onset of diabetes, these differences diminish as the rectification and inward conductance of the proximal K(IR) current increase. These diabetes-induced changes were reversed by an inhibitor of polyamine synthesis and could be mimicked by spermine, whose concentration is elevated in the diabetic eye. Hence, spermine is a candidate for mediating the effect of diabetes on the function of microvascular K(IR) channels. In addition, our findings raise the possibility that functional changes in K(IR) channels contribute to blood flow dysregulation in the diabetic retina.

Figure 1. A vascular complex freshly isolated from the retina of an adult rat

A, low magnification photomicrograph of a smooth muscle-encircled vessel and pericyte-containing microvessels that branch from this arteriole. B, enlarged view of a portion of the proximal pericyte-containing microvasculature. Arrowheads show the pericyte somas. C, enlarged view of a portion of the distal pericyte-containing microvasculature. Arrowheads show the pericyte somas. As discussed in the text, the density of abluminal pericytes is less at distal microvascular sites.

Figure 2. Effect of various concentrations of barium on the I–V relations determined from perforated-patch recordings from a pericyte located in the distal portion of the retinal microvasculature

To the left of each plot is shown the concentration of barium in the perfusate, which was solution C (10 mm K⁺) supplemented with 0–2 mm BaCl₂. Inset, current traces generated in the absence or presence of 2 mm BaCl₂; also shown is the stimulus protocol. B, relationship between the barium concentration and the amplitude of the barium-sensitive current, which was measured in distally located pericytes held −45 mV from the equilibrium potential of potassium (E_K). The amplitude of current inhibited by 2 mm BaCl₂ was defined as the maximum barium-sensitive current. Each point represents one observation; data are from six distally located pericytes each of which was exposed to multiple concentrations of barium.

Figure 3. Effect of the extracellular potassium concentration on the currents recorded from pericytes located at distal sites in the retinal microvasculature

A, current densities of the barium-sensitive conductance in distal pericytes located on isolated microvessels perfused with solution B (3 mm K⁺; n = 4), solution C (10 mm K⁺; n = 10) or solution D (97.5 mm K⁺; n = 10). The membrane capacitance, which was calculated as detailed in the Methods, was 274 ± 52 pF (n = 4), 249 ± 10 pF (n = 10) and 64 ± 22 pF (n = 10) in solutions B, C and D, respectively. For each of the potassium concentrations tested, the equilibrium potential for potassium (E_K) is shown. B, effect of increasing extracellular K⁺ from 3 mm (solution B; ▪) to 10 mm (solution C; ○) on the I–V relations of a distal pericyte.

Figure 4. Effects of extracellular barium on currents recorded from pericytes located at proximal sites in the pericyte-containing retinal microvasculature

A, I–V relations of currents during perfusion of solution C (10 mm K⁺) in the absence (○) and presence (▪) of 2 mm BaCl₂. Upper inset, current traces from which the I–V plots were generated. Also shown is the stimulus protocol. Lower inset, plot of the difference between the I–V curves. B, relationship between the barium concentration and the amplitude of the proximal barium-sensitive current, which was measured in proximally located pericytes held −45 mV from E_K. Data are from five proximally located pericytes each of which was exposed to multiple concentrations of barium.

Figure 5. Effect of extracellular potassium concentration on barium-sensitive conductance recorded from pericytes located at proximal sites within the retinal microvasculature

Plot shows the relationship between current density and voltage for the barium-sensitive conductances recorded in solution B (3 mm K⁺; n = 4), solution C (10 mm K⁺; n = 11) or solution D (97.5 mm K⁺; n = 7). For each of the tested potassium concentrations, the equilibrium potential for potassium (E_K) is shown. From perforated-patch recordings of distal pericytes, the calculated membrane capacitance was 220 ± 46 pF (n = 4), 325 ± 70 pF (n = 13) and 59 ± 25 pF (n = 7) in solutions B, C and D, respectively. Inset, the relationship between the square root of the extracellular potassium concentration and the current density. Current densities, which were determined at ±45 mV from E_K, are shown for inward and outward barium-sensitive conductances recorded at distal (○) and proximal (▪) sites. The P values for proximal and distal inward current densities being different were 0.032, 0.016 and 0.001 at 3 mm, 10 mm and 97.5 mm, respectively. The P values for proximal and distal outward current densities being different were 0.050, 0.026 and 0.010 at 3 mm, 10 mm and 97.5 mm, respectively.

Figure 6. Effect of diabetes on the microvascular K_IR current

I–V relations for the barium-sensitive current recorded at proximal sites of microvessels isolated from normal rats (▪; n = 7) and from streptozotocin-injected rats that were diabetic for 20 ± 3 days (○, n = 9) or 60 ± 8 days (▴, n = 5). Recordings were in solution D (97.5 mm K⁺). Inset, I–V plots for the barium-sensitive currents recorded in solution D at distal sites in microvessels from non-diabetic rats (▪, n = 10) and rats that had been diabetic for 69 ± 7 days (▿, n = 2); this duration of diabetes was not significantly (P = 0.5) different from that of the ‘60 day’ group.

Figure 7. Effects of spermine and DFMO on the proximal K_IR current

A, I–V relations for the proximal barium-sensitive current in the absence (▪; n = 7) and presence (○; n = 6) of 5 mm spermine in solution D (97.5 mm K⁺); in the spermine group, microvessels were exposed to this polyamine for ≥ 15 min. Inset, I–V plots of the proximal barium-sensitive current in solution D supplemented with quercetin (100 μm) minus (▪; n = 2) or plus 5 mm spermine (○; n = 4). I–V relations were generated after 15–20 min of exposure to quercetin; when used, spermine was in the perfusate for ≥ 15 min. In proximal pericytes, the barium-sensitive currents recorded in the absence or presence of quercetin did not have significantly (P = 0.6) different inward conductances or rectification quotients. B, I–V relations of the barium-sensitive current recorded at proximal sites in retinal microvessels that were either (1) isolated from diabetic rats (▴; n = 9; 86 ± 11 days of diabetes) and assayed in solution D (97.5 mm K⁺), or (2) isolated from diabetic rats and as detailed in the text, exposed to solution D (97.5 mm K⁺) supplemented with 5 mm DFMO (▪; n = 4; 88 ± 9 days of diabetes), or (3) isolated from non-diabetic rats (○; n = 7; assayed in solution D). The duration of diabetes for groups (1) and (2) was not significantly (P = 0.9) different.