Impaired small-conductance Ca2+-activated K+ channel-dependent EDHF responses in Type II diabetic ZDF rats

Abstract

We have examined the relative contributions of small- and intermediate-conductance Ca(2+)-activated K(+) channels (SK(Ca) and IK(Ca)) to the endothelium-derived hyperpolarizing factor (EDHF) pathway response in small mesenteric arteries of Zucker Diabetic Fatty (ZDF) rats, before and after the development of Type II diabetes, together with Lean controls. Smooth muscle membrane potential was recorded using sharp microelectrodes in the presence of 10 microM indomethacin plus 100 microM N(omega)-nitro-L-arginine. SK(Ca) was selectively inhibited with 100 nM apamin, whereas IK(Ca) was blocked with 10 microM TRAM-39 (2-(2-chlorophenyl)-2,2-diphenylacetonitrile). Resting membrane potentials were similar in arteries from 17- to 20-week-old control and diabetic rats (approximately -54 mV). Responses elicited by 1 and 10 microM acetylcholine (ACh) were significantly smaller in the diabetic group (e.g. hyperpolarizations to -69.5 +/- 0.8 mV (ZDF; n = 12) and -73.2 +/- 0.6 mV (Lean; n = 12; P < 0.05) evoked by 10 microM ACh). The IK(Ca)-mediated components of the ACh responses were comparable between groups (hyperpolarizations to approximately -65 mV on exposure to 10 microM ACh). However, SK(Ca)-mediated responses were significantly reduced in the diabetic group (hyperpolarizations to -63.1 +/- 1.0 mV (ZDF; n = 6) and -71.5 +/- 1.2 mV (Lean; n = 6; P < 0.05) on exposure to 10 microM ACh. Impaired ACh responses were not observed in arteries from 5- to 6-week-old (pre-diabetic) animals. SK(Ca) subunit mRNA expression was increased in the diabetic group. The EDHF pathway, especially the SK(Ca)-mediated response, is impaired in Type II diabetic ZDF rats without a reduction in channel gene expression. These results may be particularly relevant to the microvascular complications of diabetes. The functional separation of SK(Ca) and IK(Ca) pathways is discussed.

Figure 1

Effects of IbTX, ChTX and TRAM-39 on 1-EBIO-stimulated outward currents in BAE cells. Representative traces (a) and mean data measured at +50 mV (b) showing effects of cumulative addition of 100 nM IbTX and then 100 nM ChTX on 600 μM 1-EBIO (EBIO)-stimulated whole-cell currents in BAE cells (n=3). Mean data (c) showing effects of TRAM-39 (molar concentrations indicated) on EBIO-evoked outward currents measured at +50 mV (n=3). Outward currents under control (Con) conditions and in the presence of DMSO vehicle (D) before addition of EBIO are also given. ^*P<0.05 compared to EBIO.

Figure 2

ACh- and 1-EBIO-evoked responses in arteries from Old Lean and ZDF rats. Membrane potential (E_m) recordings obtained from smooth muscle impalements using sharp microelectrodes are shown. Representative traces (top) and mean data (bottom; bar top represents mean membrane potential before response, whereas bar bottom represents the mean maximum change in membrane potential during response) showing responses elicited by 600 μM 1-EBIO, 1 μM ACh and 10 μM ACh. ^*P<0.05 Lean compared to ZDF.

Figure 3

SK_Ca- and IK_Ca-mediated components of ACh- and 1-EBIO-elicited responses in arteries of Old Lean and ZDF rats. Membrane potential (E_m) recordings obtained from smooth muscle impalements using sharp microelectrodes in the presence of 100 nM apamin (a) or 10 μM TRAM-39 (b). Representative traces (top) and mean data (bottom; bar top represents mean membrane potential before response, whereas bar bottom represents mean maximum change in membrane potential during response) showing responses evoked by 600 μM 1-EBIO, 1 μM ACh and 10 μM ACh. ^*P<0.05 Lean compared to ZDF.

Figure 4

Quantification of SK3 and IK1 subunit mRNA expression by real-time RT–PCR in the arteries of Old Lean and ZDF rats. Specificity of reactions demonstrated by (a) ethidium bromide-stained agarose gels (products amplified from vector standard (V) and artery cDNA (RT)) and (b) melting curve analysis (plots of first negative derivative of fluorescence (−dF/dT) versus temperature for all artery cDNA reactions). (c) Standard curves (threshold cycle (C_T) versus log copies) for SK3 and IK1 quantification showing vector standards (black circles) and artery cDNA samples (grey circles). (d) Mean data obtained from artery cDNA samples. ^*P<0.05 Lean versus ZDF.