Diabetes downregulates large-conductance Ca2+-activated potassium beta 1 channel subunit in retinal arteriolar smooth muscle

Abstract

Retinal vasoconstriction and reduced retinal blood flow precede the onset of diabetic retinopathy. The pathophysiological mechanisms that underlie increased retinal arteriolar tone during diabetes remain unclear. Normally, local Ca(2+) release events (Ca(2+)-sparks), trigger the activation of large-conductance Ca(2+)-activated K(+)(BK)-channels which hyperpolarize and relax vascular smooth muscle cells, thereby causing vasodilatation. In the present study, we examined BK channel function in retinal vascular smooth muscle cells from streptozotocin-induced diabetic rats. The BK channel inhibitor, Penitrem A, constricted nondiabetic retinal arterioles (pressurized to 70mmHg) by 28%. The BK current evoked by caffeine was dramatically reduced in retinal arterioles from diabetic animals even though caffeine-evoked [Ca(2+)](i) release was unaffected. Spontaneous BK currents were smaller in diabetic cells, but the amplitude of Ca(2+)-sparks was larger. The amplitudes of BK currents elicited by depolarizing voltage steps were similar in control and diabetic arterioles and mRNA expression of the pore-forming BKalpha subunit was unchanged. The Ca(2+)-sensitivity of single BK channels from diabetic retinal vascular smooth muscle cells was markedly reduced. The BKbeta1 subunit confers Ca(2+)-sensitivity to BK channel complexes and both transcript and protein levels for BKbeta1 were appreciably lower in diabetic retinal arterioles. The mean open times and the sensitivity of BK channels to tamoxifen were decreased in diabetic cells, consistent with a downregulation of BKbeta1 subunits. The potency of blockade by Pen A was lower for BK channels from diabetic animals. Thus, changes in the molecular composition of BK channels could account for retinal hypoperfusion in early diabetes, an idea having wider implications for the pathogenesis of diabetic hypertension.

Figure 1. Physiological significance of BK channels in retinal arterioles

Top panel, representative photomicrographs of a pressurized retinal arteriole (70mmHg) before and 10-min after the application of 100nmol/L Pen A. Bottom panel, mean ± SEM of the Pen A-induced constriction in non-diabetic retinal arterioles (n=8 vessels, p<0.05; paired t-test).

Figure 2. Caffeine-induced BK currents recorded from retinal VSMCs of non-diabetic and diabetic animals

(A) Left, whole-cell records showing caffeine-induced outward currents in a non-diabetic vessel held at a range of potentials. The vessel was bathed in low Cl^- Hanks’ solution with 1mmol/L 9AC to block Cl_Ca currents. Right, the BK channel inhibitor, Pen A (100nmol/L), completely abolished the caffeine-induced currents. (B) Typical traces showing caffeine-induced BK currents at -80, 0, +40 and +80 mV in non-diabetic and diabetic retinal arterioles. (C) Summary current-voltage relationships for the caffeine-induced BK currents in non-diabetic (for each point, n=5-13) and diabetic (n=7-19) vessels.

Figure 3. Caffeine-evoked global [Ca²⁺]_i transients and Cl_Ca currents in non-diabetic and diabetic retinal VSMCs

(A) Left, time-course record showing the effects of 10mmol/L caffeine on global [Ca²⁺]_i in a non-diabetic retinal arteriole segment. Right, mean data showing that caffeine-induced [Ca²⁺]_i transients were unaffected by diabetes. (B) Left, whole-cell currents elicited by 10 mmol/L caffeine in a non-diabetic vessel held at a range of test potentials and bathed in normal Hanks’ solution containing the BK channel antagonist, Pen A (100nmol/L). Right, the Cl_Ca channel inhibitor, 9AC (1mmol/L), completely blocked the caffeine-evoked transient inward and outward currents. (C) Original records showing caffeine-induced Cl_Ca currents at -80, 0 and +80mV in non-diabetic and diabetic vessels. (D) Plot showing the mean peak current density against voltage for the caffeine-evoked Cl_Ca currents in non-diabetic (n=12) and diabetic (n=10) retinal VSMCs.

Figure 4. STOC activity is reduced but Ca²⁺ sparks are greater in retinal VSMCs from diabetic animals

(A) Top panel, whole-cell recordings of STOC activity in a non-diabetic and diabetic vessel at a holding potential of +40mV. Bottom panel, graph showing the mean integrated current density versus voltage for STOCs from non-diabetic (n=8) and diabetic (n=8) retinal arterioles. (B) Top panel, line-scan image recorded from a non-diabetic retinal VSMC showing two consecutive Ca²⁺ sparks originating from the same Ca²⁺ spark site. The graph below plots the fractional fluorescence change (F/F₀) for this panel. Bottom panel, line scan image and graph on slower time scales from another non-diabetic cell in which Ca²⁺ sparks amalgamate to produce a cell-wide global Ca²⁺ oscillation. (C) Representative line-scan images of basal Ca²⁺ sparks in non-diabetic and diabetic retinal VSMCs. Bottom, average temporal profile for each spark has been plotted and the traces superimposed.

Figure 5. Diabetes does not affect BK channel density

(A) Histogram showing relative BKα transcript expression in non-diabetic and diabetic retinal arterioles as determined by quantitative PCR. Amplifications were performed in triplicate. 9 non-diabetic and 9 diabetic rats were used in total and RNA was isolated from 15-25 retinal arterioles collected from 3 non-diabetic and 3 diabetic animals per replicate. BKα transcript expression was normalized to β-actin. (Bi) Family of whole-cell voltage clamp currents evoked in a non-diabetic retinal VSMC by voltage steps ranging between -100 mV to +100mV from an initial holding potential of -80mV in the presence of 9AC (1 mmol/L) and 4AP (10mmol/L). (Bii) The voltage-dependent current was abolished by the BK channel inhibitor, Pen A (100nmol/L; % change, -99.3 ± 1.1%; p<0.05; n=4), but was unaffected by (Biii) the removal of extracellular Ca²⁺ (2.5± 8.5%; p>0.05; n=4), (Biv) the L-type Ca²⁺ channel inhibitor, nifedipine (10μmol/L; -1.6 ± 17.2%; p>0.05 n=5) and (Bv) the RyR antagonist tetracaine (100μmol/L; 3.4 ± 15.2%; p>0.05; n=5). Responses were constant across the full voltage range. For clarity current records are presented for single steps between -80 to +80mV. Dashed lines: zero current (C) Average peak current density as a function of voltage for the voltage-activated BK current in non-diabetic (n=13) and diabetic (n=13) vessels.

Figure 6. Ca²⁺ sensitivity of single BK channels is reduced in diabetes

(A) Representative single BK channel records in inside-out patches (holding potential +80mV) from non-diabetic and diabetic VSMCs exposed to increasing [Ca²⁺]. (B) Summary data of the mean ± SEM P_o at the five Ca²⁺ concentrations tested. Non-diabetic, n=8-11; diabetics, n=8-11. Curves are fitted with the Hill equation as described in the supplementary methods. Fit parameters are as follows: non-diabetic (Kd = 0.86 μmol/L, Hill slope 1.1), diabetic (Kd = 1.9 μmol/L, Hill slope 0.97). (C) P_o-V relations determined at 10μmol/L Ca²⁺. Non-diabetic, n=2-11; diabetics, n=1-11. Curves are Boltzmann fits with the following parameters: non-diabetic (V_1/2 = -59.7 mV); diabetic (V_1/2 = 57.9 mV).

Figure 7. BKβ1 subunit expression and function

(A) Downregulation of BKβ1 mRNA in retinal VSMC cells from diabetic arterioles. BKβ1 expression in diabetic arterioles is presented relative to non-diabetic vessels. Amplifications were performed in triplicate (same samples as for Fig 5A) and normalized as described for BKα transcripts (B) Left, confocal images of non-diabetic and diabetic retinal arterioles embedded within retinal flatmount preparations and labeled with anti-BKβ1 Ab (green) and propidium iodide (red: nuclear label). Labeling of the circular smooth muscle is reduced in the tissue from the diabetic animal. Right, summary data showing statistically significant reduction in anti-BKβ1 fluorescence for diabetic samples (n=6 retinas, 30 vessels) relative to non-diabetics (n=6 retinas, 25 vessels). (C) Sensitivity of single BK channels in inside out patches to 1μmol/L tamoxifen (holding potential +80mV; 1μmol/L free [Ca²⁺]) from non-diabetic and diabetic retinal VSMCs. Right, summary data showing the differential effects of tamoxifen on the P_o of single BK channels from non-diabetic (n=7) and diabetic (n=8) vessels. (D) Pharmacology of single BK channels from non-diabetic (n=5) and diabetic (n=9) retinal VSMCs exposed to Pen A. Mean data is expressed as the % inhibition of P_o after 5-min of exposure to 100nmol/L Pen A.