Elevated Ca²⁺ sparklet activity during acute hyperglycemia and diabetes in cerebral arterial smooth muscle cells

Abstract

Ca⁺ sparklets are subcellular Ca²⁺ signals produced by the opening of L-type Ca²⁺ channels (LTCCs). In cerebral arterial myocytes, Ca²⁺ sparklet activity varies regionally, resulting in low and high activity, “persistent” Ca²⁺ sparklet sites. Although increased Ca²⁺ influx via LTCCs in arterial myocytes has been implicated in the chain of events contributing to vascular dysfunction during acute hyperglycemia and diabetes, the mechanisms underlying these pathological changes remain unclear. Here, we tested the hypothesis that increased Ca²⁺ sparklet activity contributes to higher Ca²⁺ influx in cerebral artery smooth muscle during acute hyperglycemia and in an animal model of non-insulin-dependent, type 2 diabetes: the dB/dB mouse. Consistent with this hypothesis, acute elevation of extracellular glucose from 10 to 20 mM increased the density of low activity and persistent Ca²⁺ sparklet sites as well as the amplitude of LTCC currents in wild-type cerebral arterial myocytes. Furthermore, Ca²⁺ sparklet activity and LTCC currents were higher in dB/dB than in control myocytes. We found that activation of PKA contributed to higher Ca²⁺ sparklet activity during hyperglycemia and diabetes. In addition, we found that the interaction between PKA and the scaffolding protein A-kinase anchoring protein was critical for the activation of persistent Ca²⁺ sparklets by PKA in cerebral arterial myocytes after hyperglycemia. Accordingly, PKA inhibition equalized Ca²⁺ sparklet activity between dB/dB and wild-type cells. These findings suggest that hyperglycemia increases Ca²⁺ influx by increasing Ca²⁺ sparklet activity via a PKA-dependent pathway in cerebral arterial myocytes and contributes to vascular dysfunction during diabetes.

diabetes is associated with an increase in vascular tone that contributes to a higher propensity for developing multiple pathological conditions including hypertension, stroke, renal failure, coronary artery disease, and retinal degeneration (1, 9, 10, 12, 16, 17, 21, 25, 48, 58). Recent studies suggest that these pathological changes in vascular dysfunction during acute hyperglycemia and diabetes are produced, at least in part, by increased contraction of the smooth muscle cells lining the walls of cerebral, mesenteric, retinal, and coronary arteries as well as skeletal arterioles due to elevated intracellular Ca²⁺ concentration ([Ca²⁺]_i) (4, 5, 7, 25, 35, 48, 49). At present, however, the cellular and molecular mechanisms underlying this increase in [Ca²⁺]_i are unclear.

Using conventional patch-clamp electrophysiological approaches, several groups have suggested that a cell-wide increase in L-type Ca²⁺ channel (LTCC) activity is a likely culprit for elevated [Ca²⁺]_i in arterial smooth muscle during diabetes (5, 35, 48, 52). However, optical recordings of Ca²⁺ influx events via LTCCs (called “Ca²⁺ sparklets”) have revealed an unexpected feature of these channels: their activity varies within the surface membrane of multiple cell types (29–32, 43, 47). There are two types of Ca²⁺ sparklet sites; low and high activity, “persistent” Ca²⁺ sparklet sites. Activation of protein kinase Cα (PKCα) induces persistent Ca²⁺ sparklet activity. At the physiological membrane potential of −40 mV, and with 2 mM external Ca²⁺, low activity and persistent Ca²⁺ sparklets contribute to Ca²⁺ influx and therefore to the regulation of [Ca²⁺]_i in arterial myocytes (2).

Although Ca²⁺ sparklets have not been examined during acute hyperglycemia and in diabetic smooth muscle, several lines of evidence suggest that an increase in low and persistent Ca²⁺ sparklet activity could contribute to higher Ca²⁺ influx into cerebral arterial myocytes during the development of diabetes. First, myogenic tone, which requires Ca²⁺ influx via LTCCs, is increased after acute hyperglycemia and diabetes in cerebral arteries (10, 12, 17, 25, 44, 58). Second, PKC and PKA activity, both of which increase LTCC activity, is increased in smooth muscle during diabetes (13, 26, 48). Third, Ca²⁺ influx through LTCCs is higher in diabetic than in control smooth muscle (5, 35, 52).

The goal of the present study was to test the hypothesis that increased low and persistent Ca²⁺ sparklet activity specifically contributes to higher Ca²⁺ influx and intracellular Ca²⁺ in cerebral artery smooth muscle cells during acute hyperglycemia and in an animal model of non-insulin-dependent, type 2 diabetes, the dB/dB mouse. We focused on cerebral arteries because the mechanisms regulating Ca²⁺ influx via LTCCs in cerebral arterial myocytes during diabetes have not been investigated. Our results are consistent with a model in which hyperglycemia increases Ca²⁺ influx into arterial myocytes by increasing low and persistent Ca²⁺ sparklet activity in arterial smooth muscle cells. Our data suggest that activation of PKA, not PKCα, contributes to higher persistent Ca²⁺ sparklet activity during hyperglycemia and diabetes. Furthermore, we found that a scaffolding protein A-kinase anchoring protein (AKAP) was critical for the local activation of persistent Ca²⁺ sparklets by PKA during acute hyperglycemia and diabetes. Together, these results support the hypothesis that an increase in low activity and persistent Ca²⁺ sparklet activity may be an early, critical event in the pathway leading to vascular dysfunction during diabetes.

MATERIALS AND METHODS

Isolation of cerebral arterial myocytes.

We used Sprague-Dawley rats as well as wild-type (WT), PKCα-knockout (PKCα^−/−) (8), diabetic dB/dB, and control BKS.Cg-m^+/+ mice. Animals were euthanized with a lethal intraperitoneal dose of pentobarbital sodium (250 mg/kg for mice and 150 mg/kg for rats), as approved by the University of Washington Institutional Animal Care and Use Committee. Smooth muscle cells were dissociated from cerebral arteries using standard enzymatic techniques described elsewhere (3). After dissociation, cells were maintained in a nominally Ca²⁺-free Ringer solution until used. Thapsigargin (1 μM) was included in all solutions used to record Ca²⁺ sparklets to eliminate Ca²⁺ release from intracellular Ca²⁺ stores during experimentation.

[Ca²⁺]_i and patch-clamp electrophysiology.

Global, cell-wide [Ca²⁺]_i was recorded as described previously (2). Briefly, global [Ca²⁺]_i was imaged in isolated arterial smooth muscle cells loaded with the membrane-permeable acetoxymethyl-ester form of Fluo-4 using a Nikon swept field confocal system coupled to a Nikon TE300 inverted microscope equipped with a Nikon ×60 lens (numerical aperture = 1.4). Background-subtracted images were normalized by dividing the fluorescence intensity of each pixel (F) for the average resting fluorescence intensity (F₀) of a confocal image to generate F/F₀ images.

Membrane currents were recorded with an Axopatch 200B amplifier using the conventional whole cell patch-clamp configuration. During experiments, cells were continuously superfused with a solution containing (in mM) 120 NMDG, 5 CsCl, 1 MgCl₂, 10 glucose, 10 HEPES, and 20 CaCl₂ adjusted to pH 7.4. Pipettes were filled with a solution composed of (in mM) 87 Cs-aspartate, 20 CsCl, 1 MgCl₂, 5 MgATP, 10 HEPES, 10 EGTA, and adjusted to pH 7.2 with CsOH. A voltage error of 10 mV attributable to liquid junction potential of these solutions was corrected offline. Whole cell LTCC currents (I_Ca) were evoked by applying a depolarizing pulse of 200 ms from the holding potential of −70 mV to +30 mV. I_Ca was measured as the difference between the peak and the sustained current at the end of the 200-ms pulse. Currents were sampled at 20 kHz and low pass-filtered at 2 kHz. Because cerebral arterial myocytes do not express functional voltage-gated T-type Ca²⁺ channels and I_Ca in these cells were blocked by dihydropyridine antagonists (41, 57), the voltage-gated, time-dependent currents evoked under these experimental conditions (i.e., Na⁺- and K⁺-free external solution) are produced by LTCCs.

Total internal reflection fluorescence microscopy.

Ca²⁺ sparklets were recorded in patch-clamped (whole cell configuration) cerebral arterial myocytes as previously described (2). Briefly, we used a through-the-lens total internal reflection fluorescence (TIRF) microscope built around an inverted Olympus IX-71 microscope equipped with an Olympus PlanApo (×60, numerical aperture = 1.45) oil-immersion lens and an Andor iXON EMCCD camera (South Windsor, CT). To image changes in [Ca²⁺]_i, we added 200 μM of the penta-potassium salt of the Ca²⁺ indicator fluo-5F to the patch pipette solution. Images were acquired at 100–300 Hz. As before (29, 30), we determined the activity of Ca²⁺ sparklets by calculating the nP_s of each Ca²⁺ sparklet site, where n is the number of quantal levels and P_s is the probability that a quantal Ca²⁺ sparklet event is active. All data sets were submitted to a normality test. Because Ca²⁺ sparklet activity has a bimodal distribution, Ca²⁺ sparklet sites were grouped into three categories: silent (by default has an nP_s of 0), low (nP_s between 0 and 0.2), and high (nP_s > 0.2). A detailed description of this analysis is given by Navedo et al. (30). Furthermore, readers are referred to a set of step-by-step video tutorials describing our Ca²⁺ sparklets analysis in our laboratory webpage (http://web.mac.com/fernando_santana/Lab_website/Home.html).

Background fluorescence was subtracted from the total fluorescence signal of fluo-5F-loaded arterial myocytes. Fluo-5F fluorescence values were then converted to Ca²⁺ concentration units using the “F_max” equation (24),

$$\left[\mathrm{C}{a}^{2+}\right]=K_d\frac{F/F_{\max }-1/R_f}{1-F/F_{\max }}$$

as previously described (2). Briefly, F is fluorescence, F_max is the fluorescence intensity of fluo-5F in the presence of saturating free Ca²⁺, K_d is the dissociation constant of (fluo-5F = 1,280 nM), and R_f (fluo-5F = 286) is the indicator's F_max/F_min ratio. F_min is the fluorescence intensity of the indicator in a solution where the Ca²⁺ concentration is 0. K_d and R_f values were determined in vitro using standard methods and are similar to those reported by others (56). F_max was determined at the end of each experiment by exposing cells to the Ca²⁺ ionophore ionomycin (10 μM) and 20 mM external Ca²⁺.

For these experiments, Ca²⁺ sparklets were recorded in cells held at the hyperpolarized potential of −70 mV and 20 mM Ca²⁺ for two reasons. First, we wanted to investigate whether the size of quantal Ca²⁺ sparklets was altered during acute hyperglycemia and diabetes. Unfortunately, quantal Ca²⁺ sparklets are very difficult to detect with 2 mM external Ca²⁺ (29). Second, we wanted to maximize our ability to determine even small changes in Ca²⁺ sparklet activity during these pathological conditions. This required that we recorded Ca²⁺ sparklet events with broad range of amplitudes. Note that with 2 mM external Ca²⁺, most Ca²⁺ sparklets have amplitudes close to or at our amplitude detection threshold of 18 nM, suggesting the presence of frequent subthreshold Ca²⁺ sparklets (29). Thus, using 2 mM external Ca²⁺, although physiologically relevant, may have led to underestimation of Ca²⁺ sparklet activity because low-amplitude events may fall below our detection threshold.

Plasma glucose.

Plasma glucose levels were measured from tail vein samples using a commercially available kit (One Touch UltraMini, LifeScan, Milpitas, CA) according to manufacturer instructions.

Chemicals and statistical analysis.

All chemicals were from Sigma-Aldrich (St. Louis, MO) unless stated otherwise. Normally distributed data are presented as means ± SE. Comparison of the entire nP_s data set and nonnormally distributed data was performed using nonparametric data analyses (Mann-Whitney test). Comparison between low and high nP_s groups and two-sample comparisons were performed using parametric data analyses (two-tailed, Student's t-test). P < 0.05 was considered significant.

RESULTS

Acute hyperglycemia increases L-type Ca²⁺ currents and global [Ca²⁺]_i in cerebral arterial myocytes.

We measured plasma glucose concentration in control, nondiabetic mice and in an animal model of type 2 diabetes, the dB/dB mouse. Plasma glucose concentration was 9.1 ± 0.3 mM (n = 4) and 22.8 ± 2.0 mM (n = 4) in control and dB/dB mice, respectively. These data are consistent with previous studies suggesting that plasma glucose levels range between 6 and 10 mM in nondiabetic animals and about 20 mM in humans and animal models of type 2 diabetes (21, 27).

Next, we investigated the effects of acute increases in extracellular d-glucose concentration on global [Ca²⁺]_i in arterial myocytes dissociated from cerebral arteries from nondiabetic, control mice. [Ca²⁺]_i was imaged in cells loaded with the fluorescent Ca²⁺ indicator Fluo-4 using a confocal microscope (Fig. 1). Increasing extracellular d-glucose from 10 to 20 mM evoked an increase in global [Ca²⁺]_i (Fig. 1A). We expanded these experiments by examining the effects of a broader range of d-glucose concentrations (5 to 40 mM) on global [Ca²⁺]_i in cerebral arterial myocytes. Note that increasing d-glucose concentration from 5 mM ([Ca²⁺]_i = 1.1 ± 0.02 F/F₀) to 10 mM ([Ca²⁺]_i = 1.3 ± 0.07 F/F₀) did not increase global [Ca²⁺]_i (n = 6 cells; P > 0.05). As shown in Fig. 1A, an elevation in external d-glucose concentration from 10 mM to 20 mM ([Ca²⁺]_i = 1.6 ± 0.1 F/F₀) evoked a significant increase in global [Ca²⁺]_i (P < 0.05). However, we found that increasing external d-glucose from 20 mM to 40 mM ([Ca²⁺]_i = 1.8 ± 0.2 F/F₀) did not translate into a further increase in [Ca²⁺]_i (P > 0.05). Indeed, fitting this relationship with a sigmoidal function revealed that the d-glucose concentration that evoked a half-maximal increase (i.e., EC₅₀) in [Ca²⁺]_i was 15 mM.

Fig. 1.

An elevation in extracellular d-glucose increases global intracellular Ca²⁺ concentration ([Ca²⁺]_i) and L-type calcium channel current (I_Ca) in rat cerebral arterial myocytes. A: representative trace of global [Ca²⁺]_i in arterial myocyte exposed to 10 mM d-glucose and after application of 20 mM d-glucose (as indicated by the arrow; n = 9 cells). B: concentration-response curve for d-glucose-induced changes in global [Ca²⁺]_i in cerebral arterial myocytes (n = 6 cells). Solid line represents best-fit curve to data determined by a least-squares method using a sigmoidal dose-response equation: y = {A₁ + (A₂ − A₁)/1 + 10^{[(logEC50 − X) × k]}}, where A₁ (0.97), A₂ (1.65), EC₅₀ (15.16), X (1.20), and k (2.80) are the initial value, final value, the d-glucose concentration at which 50% of the [Ca²⁺]_i response was observed, and the slope factor. Inset: plot of the mean ± SE of global [Ca²⁺]_i in myocytes exposed to 20 mM d-glucose or 20 mM l-glucose. C: representative I_Ca recordings under control conditions and after acute application of 20 mM d-glucose (n = 7 cells). The graph at right plots the mean ± SE of the amplitude of I_Ca (at +30 mV) before and after application of 20 mM d-glucose. *P < 0.05.

As a control for these experiments, we examined the effects of the nonmetabolizable levo enantiomer of glucose l-glucose and the nonpermeable sugar mannitol on global [Ca²⁺]_i. Unlike d-glucose, application of 20 mM l-glucose (inset in Fig. 1B) or 20 mM mannitol (not shown) did not increase [Ca²⁺]_i in arterial myocytes (n = 8 cells per experimental group; P < 0.05). Together with the plasma concentration data described above, these findings suggest that changes in external d-glucose between 10 and 20 mM—not changes in the osmolarity of the extracellular solution—are physiologically relevant and translate into sustained elevations in global [Ca²⁺]_i in cerebral arterial smooth muscle.

LTCCs are important regulators of global [Ca²⁺]_i in cerebral arterial myocytes (20, 41). Thus, to investigate the mechanisms by which d-glucose increases [Ca²⁺]_i, we examined the effects of increasing d-glucose from 10 (i.e., control) to 20 mM on I_Ca in voltage-clamped arterial myocytes (Fig. 1C). In these experiments, I_Ca was evoked by 200-ms voltage steps from the holding potential of −70 mV to the test potential of +30 mV. Currents were recorded at multiple time points until a steady-state change in whole cell I_Ca in the presence of 20 mM d-glucose was observed (typically 5 min after exposure to this solution). Consistent with the [Ca²⁺]_i data described above, we found that increasing extracellular d-glucose from 10 to 20 mM increased I_Ca amplitude by ∼66% (n = 7; P < 0.05). These findings suggest that acute hyperglycemia increases [Ca²⁺]_i, at least in part, by increasing I_Ca in cerebral arterial myocytes.

Acute elevation of extracellular d-glucose increase Ca²⁺ sparklets in cerebral arterial myocytes.

We investigated the mechanisms by which an elevation in d-glucose increases I_Ca in voltage-clamped cerebral arterial myocytes. To do this, we examined the elementary events of Ca²⁺ influx via LTCCs in these cells: Ca²⁺ sparklets (Fig. 2) (29, 50). We determined Ca²⁺ sparklet density, amplitude, and activity before and after the application of an extracellular solution containing 20 mM d-glucose. As described here in detail in materials and methods and elsewhere (29, 30), Ca²⁺ sparklet activity was quantified as nP_s, where n and P_s are the number of quantal levels and probability of sparklet occurrence, respectively.

Fig. 2.

An elevation in extracellular d-glucose increases Ca²⁺ sparklet activity in rat cerebral arterial myocytes. A: representative total internal reflection fluorescence (TIRF) image of a cerebral arterial myocyte (left). Traces (right) are the time course of [Ca²⁺]_i in the sites indicated by the green circles before and after exposure to 20 mM d-glucose. B: Ca²⁺ sparklet density before and after 20 mM d-glucose (n = 7 cells) or 20 mM l-glucose (n = 5 cells). Ct, control. C: amplitude histogram of Ca²⁺ sparklets before and after 20 mM d-glucose. The black lines are the best fit to the data with a multicomponent Gaussian function

$$N={\displaystyle \sum _{j=1}^n{a}_{\mathrm{j}}}\times \exp [-\frac{{({[\mathrm{C}{\mathrm{a}}^{2+}]}_{\mathrm{i}}-jq)}^2}{2jb}]$$

where a and b are constants and q is the quantal unit of Ca²⁺ influx (33 and 34 nM for control and 20 mM d-glucose, respectively). D: nP_s (where n and P_s are the number of quantal levels and probability of sparklet occurrence, respectively) values for individual Ca²⁺ sparklet sites under control conditions and after application of 20 mM d-glucose or 20 mM l-glucose. The dashed line defines the threshold between low and high nP_s sites. *P < 0.05.

Consistent with our [Ca²⁺]_i and I_Ca data above, increasing extracellular d-glucose , but not l-glucose or mannitol, from 10 to 20 mM increased Ca²⁺ sparklet density nearly 1.8-fold (Fig. 2, A and B; n = 7 cells; P < 0.05). Indeed, analysis of Ca²⁺ sparklet records indicates that application of 20 mM d-glucose, but not l-glucose or mannitol, augmented total Ca²⁺ sparklet activity in cerebral arterial myocytes nearly 12-fold by increasing the activity (i.e., nP_s) of previously active sparklet sites and by activating new sites (P < 0.05; Fig. 2, A–D).

As part of this analysis, we generated an amplitude histogram of Ca²⁺ sparklet events under control conditions and after exposure to 20 mM d-glucose (Fig. 2C). As previously reported (29, 30), these Ca²⁺ sparklet amplitude histograms could be fit with a multi-Gaussian function with a quantal unit of Ca²⁺ influx of 33 and 34 nM for control and 20 mM d-glucose, respectively. These results indicate that 20 mM d-glucose increases the number of Ca²⁺ sparklets observed without changing the amplitude of elementary Ca²⁺ sparklets. Collectively, these findings suggest that elevations in extracellular d-glucose increases I_Ca and hence global [Ca²⁺]_i by increasing Ca²⁺ sparklet activity, but not the amplitude of quantal Ca²⁺ sparklet events in arterial myocytes.

Acute elevation of extracellular d-glucose increases Ca²⁺ sparklet activity by increasing PKA activity in cerebral arterial myocytes.

Increased PKC activity has been reported in smooth muscle during diabetes (13, 38, 53, 54). Indeed, recent work from our laboratory (29, 30, 32) suggested that PKCα activity is required for high activity, but not for low activity Ca²⁺ sparklets. Thus, we investigated whether PKCα was required for the increase in Ca²⁺ sparklet activity after an elevation in extracellular d-glucose from 10 to 20 mM. In these experiments, the effects of acute hyperglycemia on Ca²⁺ sparklets were examined in cerebral arterial myocytes from WT and PKCα-null (PKCα^−/−) mice.

We found that, like WT (see Fig. 2), increasing d-glucose from 10 to 20 mM increased Ca²⁺ sparklet site density (from 0.02 ± 0.01 to 0.09 ± 0.05 sparklet sites/μm²) and activity (i.e., nP_s; from 0.002 ± 0.001 to 0.29 ± 0.11) in PKCα^−/− myocytes (Fig. 3, A, C, and D) (n = 10 cells; P < 0.05). These results suggest that PKCα is not required for increased Ca²⁺ sparklet activity in cerebral arterial myocytes during acute hyperglycemia. Note, however, that a recent study suggested that high glucose increases Gαs activity, which could increase cAMP production and hence PKA activity (23). Thus, we investigated whether elevations in extracellular glucose increase Ca²⁺ sparklet activity by activating PKA in arterial myocytes. To do this, we exposed PKCα^−/− cerebral arterial myocytes to 20 mM d-glucose in the presence of the PKA inhibitor rpcAMP (100 μM). Consistent with our hypothesis, increasing d-glucose from 10 to 20 mM in the presence of rpcAMP failed to increase Ca²⁺ sparklet density or activity in PKCα^−/− myocytes (Fig. 3, B–D) (n = 10 cells; P > 0.05). This result suggests that hyperglycemia increases Ca²⁺ sparklet activity through the activation of PKA in cerebral arterial myocytes.

Fig. 3.

PKA, but not PKCα, is required for increased Ca²⁺ sparklet activity after an elevation in extracellular d-glucose in mouse cerebral arterial myocytes. A and B: TIRF images of representative PKCα^−/− arterial myocytes. The traces below the images show the time course of [Ca²⁺]_i in the sites indicated by the green circles before and after exposure to 20 mM d-glucose (n = 10 cells; A) or 20 mM d-glucose + rpcAMP (n = 10 cells; B). C and D: Ca²⁺ sparklet density (C) and plot of nP_s values (D) for individual Ca²⁺ sparklet sites in wild-type (WT; n = 7 cells) and PKCα^−/− arterial myocytes before and after exposure to 20 mM d-glucose or 20 mM d-glucose + rpcAMP. *P < 0.05.

Activation of PKA increases Ca²⁺ sparklet activity.

A testable prediction of the findings described above is that activation of PKA is sufficient to increase Ca²⁺ sparklet activity in cerebral arterial myocytes. To test this hypothesis, we examined Ca²⁺ sparklet activity in WT myocytes before and after the application of the adenyl cyclase activator forskolin (10 μM) in the presence of 10 mM d-glucose (e.g., control solution). Forskolin increases PKA activity by increasing cAMP levels. Consistent with our hypothesis, forskolin increased (P < 0.05, n = 7 cells) Ca²⁺ sparklet density (from 0.04 ± 0.01 to 0.16 ± 0.06) and activity (from 0.06 ± 0.03 to 0.83 ± 0.17) (Fig. 4), indicating that activation of PKA is sufficient to increase Ca²⁺ sparklet activity in cerebral arterial myocytes even in the presence of control d-glucose (10 mM) concentration.

Fig. 4.

PKA activation induces persistent Ca²⁺ sparklet activity in rat cerebral arterial myocytes. A: sample TIRF image of a typical WT arterial myocyte. Traces below the image show representative time course of [Ca²⁺]_i in the site indicated by the green circle before and after activation of PKA by forskolin (10 μM). B and C: Ca²⁺ sparklet density (B) and nP_s analysis of Ca²⁺ sparklet sites (C) before and after application of forskolin. *P < 0.05; n = 7 cells.

Targeting of PKA by AKAP is required for increased Ca²⁺ sparklet activity during acute hyperglycemia.

PKA is targeted to specific regions of the surface membrane of mammalian cells by the scaffolding protein AKAP (19, 55). We investigated whether targeting of PKA by AKAP is required for the increase in I_Ca and Ca²⁺ sparklet activity during acute hyperglycemia by examining the effects of increasing extracellular glucose in the presence or absence of the specific peptide inhibitor of PKA-AKAP interaction HT31 (10 μM). HT31p (10 μM; a proline-substituted derivative), which does not bind the RII subunit of PKA and hence does not prevent PKA-AKAP interactions, was used as a negative control peptide. In contrast to cerebral arterial myocytes under control conditions [I_{Ca(20 mM d-glucose)}/I_{Ca(10 mM d-glucose)} = 1.5 ± 0.1; n = 9 cells] or exposed to HT31p [I_{Ca(20 mM d-glucose)}/I_{Ca(10 mM d-glucose)} = 1.6 ± 0.1; n = 10 cells], application of 20 mM d-glucose did not increase I_Ca [Fig. 5A; I_{Ca(20 mM d-glucose)}/I_{Ca(10 mM d-glucose)} = 0.97 ± 0.05; n = 5 cells] and Ca²⁺ sparklet density or activity (Fig. 5, B–D) in the presence of HT31. These findings suggest that direct targeting of PKA by AKAP is necessary for high glucose-induced increases in Ca²⁺ sparklet activity in cerebral arterial myocytes.

Fig. 5.

An A-kinase anchoring protein (AKAP) is required for increased Ca²⁺ sparklet activity in rat cerebral arterial myocytes after an elevation in extracellular d-glucose. A: representative I_Ca recordings after acute application of 20 mM d-glucose in cells with no peptide dialyzed (n = 7 cells) and cells dialyzed with HT31 (10 μM) or HT31p (10 μM; n = 6 and 10 cells, respectively). The graph at right plots the mean ± SE of the change in I_Ca amplitude (at +30 mV) after application of 20 mM d-glucose normalized to the I_Ca obtained with 10 mM d-glucose. B: representative time courses of [Ca²⁺]_i before and after exposure to 20 mM d-glucose + HT31 (10 μM; n = 9 cells) or 20 mM d-glucose + HT31p (10 μM; n = 6 cells). C and D: Ca²⁺ sparklet density (C) and scatterplot of nP_s values (D) for individual Ca²⁺ sparklet sites before and after 20 mM d-glucose or 20 mM d-glucose + HT31. *P < 0.05.

Increased Ca²⁺ sparklet activity in cerebral arterial myocytes from an animal model of type 2 diabetes.

All the experiments described above examined the effects of acute elevations of extracellular d-glucose on I_Ca and Ca²⁺ sparklets. Thus, we investigated whether I_Ca and Ca²⁺ sparklet activity is increased in cerebral arterial myocytes from 12- to 13-wk-old animal model of type 2 diabetes: the dB/dB mouse (Fig. 6) (21). As a control, we used cerebral smooth muscle cells isolated from aged-match BKS.Cg-m^+/+ (WT) mice. Consistent with previous reports (6, 37, 45), we found that body weight (51 ± 0.4 vs. 32 ± 0.5 g; n = 4 mice) and blood glucose (9.1 ± 0.3 and 22.8 ± 2.0 mM; n = 4 mice) were greater in dB/dB than in WT mice (P < 0.05) respectively.

Fig. 6.

I_Ca and Ca²⁺ sparklet activity is increased in cerebral arterial myocytes from diabetic mice. A: representative I_Ca recordings from BKS.Cg-m^−/− (WT) and dB/dB arterial myocytes. The graph at right plots the mean ± SE of the amplitude of I_Ca (at +30 mV) from WT and diabetic arterial myocytes (n = 8 cells per group). B: sample TIRF images from WT (top) and diabetic (bottom) arterial myocytes. Traces at right show time courses of [Ca²⁺]_i in the sites indicated by the green circles from WT and diabetic arterial myocytes. C and D: Ca²⁺ sparklet density (C) and scatterplot of nP_s values (D) from Ca²⁺ sparklet sites of WT arterial myocytes (n = 13 cells) and diabetic smooth muscle cells (n = 11 cells) before and after PKA inhibition with PKAi (100 nM). *P < 0.05.

I_Ca was evoked using a voltage protocol similar to one implemented in Fig. 1A. I_Ca was ∼50% larger in diabetic dB/dB myocytes (1.65 ± 0.15 pA/pF, n = 7 cells) than in WT myocytes (0.75 ± 0.11 pA/pF, n = 7 cells; P < 0.05). In addition, we found that Ca²⁺ sparklet density and activity were higher (P < 0.05) in dB/dB (0.11 ± 0.03 sites/μm²; nP_s = 0.32 ± 0.05; n = 11 cells) than in WT (0.04 ± 0.02 sites/μm²; nP_s = 0.13 ± 0.08; n = 13 cells) myocytes (Fig. 6, B–D). Consistent with the hypothesis that PKA is required for increase Ca²⁺ sparklet activity during hyperglycemia and diabetes, inhibition of this kinase with the specific inhibitor PKAi (100 nM) prevented the increase in Ca²⁺ sparklet density and activity observed in dB/dB arterial myocytes (P > 0.05; Fig. 6, C–D).

DISCUSSION

The data in this study support a new model for increased Ca²⁺ influx into arterial myocytes from cerebral arteries during acute hyperglycemia and diabetes. In this model, elevations in extracellular d-glucose from 10 mM to ≥ 20 mM increase the activity of PKA, which translates into an increase in the density and activity of low and persistent Ca²⁺ sparklet sites in specific regions within the sarcolemma of arterial myocytes. Our data suggest that PKA targeting by an AKAP is essential for increased Ca²⁺ sparklet activity during hyperglycemia. Thus, an increase in I_Ca during acute hyperglycemia and diabetes results from an increase in low activity and persistent Ca²⁺ sparklet sites due to activation of AKAP-targeted PKA.

This is the first report suggesting that PKA—via an AKAP—can induce persistent Ca²⁺ sparklet activity in arterial myocytes. At present, the molecular identity of the AKAP isoform(s) responsible for targeting PKA to specific regions of the sarcolemma of arterial myocytes is unclear. One potential candidate is AKAP150, which binds PKA, PKCα, and the Ca²⁺-sensitive phosphatase calcineurin (also known as protein phosphatase 2B), interacts with LTCCs (14, 19, 36), and is required for PKCα-dependent persistent Ca²⁺ sparklet activity in arterial smooth muscle (30). The functional role of AKAP150 in PKA signaling in arterial myocytes during physiological condition and diabetes is unclear, however. Future studies should address these important issues.

Multiple studies have suggested that PKA activation relaxes arterial smooth muscle by reducing global [Ca²⁺]_i. PKA accomplishes this, at least in part, by increasing the activity of large-conductance Ca²⁺-activated K⁺ (BK) channels, which hyperpolarize and therefore decrease Ca²⁺ influx through LTCCs (39, 51). Interestingly, PKA also increases LTCC activity (see Fig. 3) (15, 42). These findings raise a difficult question: how can activation of PKA increase LTCC activity and yet decrease global [Ca²⁺]_i in arterial smooth muscle? Although our study does not provide a definitive answer to this question, recent work suggesting compartmentalization of cAMP/PKA signaling in ventricular myocytes may provide insights into this conundrum (11, 18, 28). In these cells, exposing a small fraction of the sarcolemma to the β-adrenergic receptor agonist isoproterenol caused a local increase in cAMP and PKA activity. This increase in cAMP/PKA signaling was restricted to the site of application of the drug. However, exposing a similar fraction of the sarcolemma of ventricular myocytes to the PKA agonist forskolin resulted in a global, cell-wide increase in cAMP levels (18).

The consensus is that subcellular targeting of adenyl cyclases, PKA, and phosphodiesterases by AKAPs restricts cAMP/PKA signaling to the vicinity of sarcolemmal β-adrenergic receptors that are activated in ventricular myocytes (11, 18, 28). However, by entering the cell and diffusing broadly within it, forskolin causes a global increase in cAMP level that activates PKA in multiple regions within the cell. On the basis of these findings, a model has been proposed in which different PKA targets could be differentially modulated depending on the spatial and temporal characteristics of PKA activation in ventricular myocytes (11, 18, 28).

According to this model, an agonist that primarily activates PKA targeted to the sarcolemma near LTCCs in smooth muscle would increase Ca²⁺ influx and consequently global [Ca²⁺]_i in these cells. In contrast, an agonist like forskolin, which could induce a cell-wide activation of PKA, would lead to the phosphorylation of multiple proteins including phospholamban, SERCA, ryanodine receptors, BK channels, and LTCCs. In this case, although LTCCs are phosphorylated, [Ca²⁺]_i decreases presumably because the membrane hyperpolarization produced by the activation of BK channels diminishes the activity of LTCCs to a point in which Ca²⁺ entry is lower than under control conditions. Thus, the functional consequences of a specific PKA agonist would be dependent on the spatiotemporal characteristics of PKA signaling in arterial myocytes. In this model, HG-induced activation of PKA could be restricted to regions of the cell near LTCCs, thus causing increased Ca²⁺ influx.

Our observation that persistent Ca²⁺ sparklet activity is increased during acute hyperglycemia and diabetes has important implications. Persistent Ca²⁺ sparklets activate nearby calcineurin, which dephosphorylates and thereby activates the transcription factor NFATc3 in arterial myocytes during hypertension (33). Elegant work by María Gomez's group suggests that elevations in extracellular glucose activate calcineurin and NFATc3 signaling in cerebral artery smooth muscle (35). Interestingly, activation of NFATc3 can downregulate the expression of the α- and β₁-subunits of the large conductance, Ca²⁺-activated K⁺ channel and voltage-gated Kv2.1 channels (22, 33, 34). Thus it is intriguing to speculate that an increase in persistent Ca²⁺ sparklet activity contributes, at least in part, to NFATc3 activation and thus decreased K⁺ channel expression during diabetes. Future studies should examine the relationship between Ca²⁺ sparklets, NFATc3 activity, and K⁺ channel expression during diabetes.

Resistance arteries play a crucial role in the regulation of blood pressure and control of tissue perfusion (20). Previous studies have shown enhanced constriction and myogenic tone in cerebral arteries during acute hyperglycemia and in animal models of diabetes (e.g., dB/dB) (10, 12, 17, 25, 44, 58). Several mechanisms may contribute to this change in vascular reactivity. Previous studies have suggested that activation of PKC in cerebral arteries underlies decreased voltage-gated K⁺ currents in their smooth muscle (40, 44) and impaired endothelium-dependent vasodilation in these arteries (10, 25, 58) during hyperglycemia and diabetes (e.g., dB/dB mouse). This is important for two reasons. First, together with our findings, it suggests that multiple kinases (i.e., PKA and PKC) are activated during hyperglycemia and diabetes. Second, in vivo, PKC, NFATc3-dependent decreases in K⁺ currents, and impaired endothelium-dependent vasodilation during hyperglycemia and diabetes would depolarize smooth muscle, which would increase low activity and persistent Ca²⁺ sparklet activity in arterial myocytes. The data in Fig. 1, A and B, provide examples in which an increase global [Ca²⁺]_i during acute hyperglycemia likely resulted from the combinatorial effects of membrane depolarization and direct LTCC activation by PKA, because the cells examined in these experiments were not voltage clamped.

An interesting observation in our study is that Ca²⁺ sparklet activity and I_Ca are higher in dB/dB than in WT arterial myocytes even in the presence of control d-glucose (10 mM) concentration. Note, however, that the differences in Ca²⁺ sparklet activity are eliminated by PKA inhibition, suggesting that increased Ca²⁺ sparklet density and activity is the result of higher PKA activity in dB/dB than in WT myocytes under these experimental conditions. Whether higher PKA activity in dB/dB than WT myocytes is the result of differential adenyl cyclase, phosphodiesterase, and/or PKA expression and activity in these cells is presently unclear.

How does an increase in extracellular glucose activate PKA in arterial myocytes? Recent studies suggest an answer to this difficult question. Research from several groups suggested that increased extracellular glucose activates Gαs, which would activate adenyl cyclase and hence the production of cAMP (23, 46). An increase in cAMP would then activate PKA.

To conclude, our data indicate that AKAP-targeted PKA plays a crucial role in the regulation of Ca²⁺ influx via LTCCs and [Ca²⁺]_i in cerebral vascular smooth muscle during hyperglycemia and diabetes. We demonstrate that increased Ca²⁺ influx during hyperglycemia and diabetes is not the result of a broad, cell-wide activation of LTCCs. Instead, our data suggest that increased Ca²⁺ influx in cerebral arterial myocytes during hyperglycemia and diabetes results primarily from increased PKA-dependent low activity and persistent Ca²⁺ sparklet activity in specific sarcolemma microdomains. Finally, our data suggest that an increase in number and probability of activation of Ca²⁺ sparklet sites—not an increase in the quantal unit of Ca²⁺ influx—underlies increased Ca²⁺ influx via LTCCs into cerebral arterial myocytes during hyperglycemia and diabetes. These increases in persistent Ca²⁺ sparklet activity could potentially modulate excitability, contraction, and gene expression in smooth muscle during diabetes.

GRANTS

This study was supported by grants from the American Heart Association (to M. F. Navedo and L. F. Santana) and National Institutes of Health (to L. F. Santana).

DISCLOSURES

No conflicts of interest are declared by the author(s).