Alternative Splicing of cGMP-Dependent Protein Kinase I in Angiotensin-Hypertension

Animal Models and Cell Isolation

All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Maryland. Angiotensin-hypertensive rats were prepared by implanting female Wistar-Kyoto (WKY) rats (220 to 300 g; Harlan Sprague-Dawley) with osmotic minipumps (Alzet 2002, Alza Corporation) that delivered 0.9% NaCl with angiotensin II (Ang; Sigma; 240 μg/kg per hour) for 4 weeks (AHR) or 8 weeks (2m-AHR). Controls were age-matched female WKY rats with no implanted pump. We also studied animals with implanted pumps delivering Ang at the same rate as in AHR that were sacrificed at 4 days (4d-Ang), before development of hypertension.¹⁸ Systolic blood pressure (mean±SE), measured in awake animals by tail-cuff plethysmography (Harvard Instruments), was 137±13, 135±15, 205±10, and 193±15 mm Hg in control rats, 4d-Ang, AHR, and 2m-AHR, respectively. Basilar arteries were harvested and processed for isolation of single cells by enzymatic digestion as previously described.^18,19⇓

Patch Clamp

Methods used for perforated patch recording of Maxi-K channels have been described.¹⁹ For inside-out patch recordings, the pipette contained (mmol/L) NaCl 140, KCl 5, MgCl₂ 2, HEPES 10, and glucose 12.5. The bath contained (mmol/L) KCl 145, K₂ATP 1, MgCl₂ 2, HEPES 10, EGTA 2.48, CaCl₂ 1, and glucose 10.

8-Br-cGMP, 8-aminophenylthio-guanosine-3′,5′-cyclic monophosphate (8-APT-cGMP), and recombinant cGKIα (catalogue No. 370650) were from Calbiochem.

Immunofluorescence

Animals were perfusion-fixed with 4% formaldehyde in PBS, and brains were processed for paraffin sectioning (4 μm) as described.¹⁸ Primary antibodies used were anti-cGKIα/β (1:1000) and anti-cGKIβ (1:2000) from Stressgene (Victoria, British Columbia).

For quantitative immunofluorescence²⁰ for cGKI, tissue sections from all animals in all experimental group were immunolabeled as a single batch. Images were collected using a Nikon Eclipse E1000 microscope and a SenSys digital camera (Photometrics) with IPLab software (version 3.06, Scanalytics) using uniform parameters of magnification and exposure. Single-plane wide-field images were deconvoluted using a point-spread function computed with microscope-specific optical parameters (Huygens Essential, SVI), and the percentage of area occupied by “bright particles” in equal-sized regions of interest within VSMC layers was computed using IPLab software.

Western Blotting and Coimmunoprecipitation

Western blots of basilar artery total protein for cGKIα/β and cGKIβ were obtained as described,¹⁸ using the antibody probes listed above. Coimmunoprecipitation of cGKI with Maxi-K (Slo) channels was carried out as described,¹⁸ using total protein pooled from 6 to 8 animals and antibody directed against mSlo (Alomone).

Isolation of Total RNA and Real-Time PCR

Quantitative PCR was performed using the real-time TaqMan-PCR method. Total RNA was isolated from basilar arteries using an RNeasy Mini Kit and an RNase-Free DNase Set (Qiagen). RNA (200 ng) was reverse-transcribed using Ominiscript RT Kit (Qiagen). A 1:10 dilution of the resulting cDNA was used as the template to quantify the relative content of mRNA by real-time PCR (Bio-Rad, iCycler iQ Real-Time PCR Detection System) using appropriate primers and SYBR Green. The following primers for real-time PCR were designed using Primer Express software (AB), based on published sequences: cGKIα (accession No. XM_219813): forward primer (bases 635 to 654), 5′-AAGACGGCAAGCATGAAGCT-3′; reverse primer (bases 706 to 684), 5′-CCCTTCTGTCCCTGTAAAGGTTT-3′; cGKIβ (accession No. XM_219812): forward primer (bases 427 to 447), 5′-CAGGGACAGTGTAGCCGAACA-3′; reverse primer (bases 495 to 476), 5′-TGCGACCCGAGTATGAGACA-3′. PCR reactions were performed with the iQ SYBR Green supermix kit (Bio-Rad). Parameters included an initial denaturation at 94°C for 180 seconds, followed by 40 cycles at 95°C for 30 seconds, 60°C for 25 seconds, 72°C for 30 seconds, and 1 cycle at 72°C for 7 minutes. Fluorescence data were acquired at the end of extension. A melt analysis was run for all products to determine the specificity of the amplification. In addition, PCR products were run on 2.5% agarose gels to confirm that correct band sizes were present. A control fragment of 18S RNA (187 bp) was amplified at the same time and used for quantification. The cycle threshold values for 18S RNA and that of the samples were measured and calculated by computer software (iCycler IQ OSS, version 3.0a, Bio-Rad).

Northern Blot Analysis

cDNA probes were produced by RT-PCR. Nucleotides 4 to 266 of cGKIα (5′-AACATGAGCGAGCTGGAGGAAGAC-3′ and 5′-GATTTGGTGAACTTCCGGAATGCC-3′) and nucleotides 1 to 472 of cGKIβ (5′-GCAGACTGGGCATGCTCAGAAGCC-3′ and 5′-TGGACTCTTGGGGTAGAAGGGCAG-3′) were amplified. Products were subcloned into pDrive Cloning vector and transcribed into RNA and were used as probes. Total RNA from basilar artery was isolated for Northern blot analysis using standard techniques. Sense probe and GAPDH were used as controls.

Data Analysis

Statistical comparisons were evaluated using either ANOVA, with Student-Newman-Keuls comparisons, or Student’s t test, as appropriate. Data are given as mean±SE.

Identification of Maxi-K Channel

The recording conditions used to measure Maxi-K channel activity were formulated to eliminate currents from other channels expressed in these native cells. Cells from both controls and AHR exhibited fluctuating outward currents (Figures 1A and 1B) that were highly sensitive to block by the classical Maxi-K channel blocker charybdotoxin (Figures 1C and 1D). Inside-out patches from control and AHR cells exhibited single-channel openings with open-channel conductances of 157±1 and 143±5 pS, respectively (Figures 1E through 1G), which were not significantly different.

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Figure 1. Maxi-K channel in basilar artery cells. A and B, Original records of Maxi-K currents in basilar artery cells from control (A) and AHR (B), recorded during 200-ms test pulses from +10 to +80 mV (10-mV increments) from holding potential of 0 mV. C and D, Maxi-K currents in basilar artery cells from control (C) and AHR (D), recorded during test pulses to +80 mV, before (CTR, AHR) and after (ChTX) addition of charybdotoxin (100 nmol/L). E and F, Single-channel recordings of Maxi-K channels in inside-out patches of basilar artery cells from control (E) and AHR (F) recorded at holding potential of 0 mV. G, Slope conductance of open-channel current of Maxi-K channels from control (empty circles) and AHR (filled circles), with pooled data (5 patches from both) showing conductance of ≈150 pS.

Nitrate Tolerance

In control cells, the NO donor sodium nitroprusside (SNP, 100 nmol/L) caused a large increase in Maxi-K channel current that reached steady state in 5 to 6 minutes (Figures 2A and 2C) and that was blocked by charybdotoxin (100 nmol/L; not shown).^13,14⇓ By contrast, when tested in cells from AHR, SNP elicited a much smaller increase in current (Figures 2B and 2C). The increase in current in response to SNP in cells from AHR and from 2m-AHR was significantly (by ANOVA, P<0.01) less than in controls (Figure 2D), consistent with NT in AHR, and indicating a stable model of hypertension.

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Figure 2. Nitrate tolerance in AHR. A and B, Maxi-K currents in basilar artery cells from control (A) and AHR (B), recorded during test pulses to +80 mV, before and after addition of 100 nmol/L SNP. C, Time course of Maxi-K channel activation by 100 nmol/L SNP in basilar artery cells from control (CTR; 8 cells) and AHR (9 cells). D, Fractional increase in Maxi-K channel current 6 minutes after addition of 100 nmol/L SNP in cells from control (CTR), AHR, and 2m-AHR (7 cells). E, Time course of Maxi-K channel activation by 100 μmol/L 8-Br-cGMP in basilar artery cells from control (CTR; 9 cells) and AHR (7 cells). F, Fractional increase in Maxi-K channel current 6 minutes after addition of 100 μmol/L 8-Br-cGMP in cells from control (CTR), AHR, and 2m-AHR (7 cells), without kinase inhibitor (empty bars) or with 1 μmol/L KT-5823 (black bar); **P<0.01. Cells for individual experiments were pooled from 3 to 5 animals.

Possible Mechanisms of NT

Concentration Response for 8-Br-cGMP

With 500 μmol/L 8-Br-cGMP, the blunted effect previously seen in AHR was eliminated, with cells from both controls and AHR showing a comparable increase in current (Figures 3A and 3B). These large increases were not blocked by KT-5720, indicating no involvement of cAK. Experiments to determine the concentration-response relationship showed that, at all but the highest concentrations used, the fractional increase in current was significantly less in AHR than in controls (Figure 3C). Fit of the data to a standard logistic equation gave EC₅₀ values of 44 and 110 μmol/L for controls and AHR, respectively, with Hill coefficients of ≈1.9 (Figure 3C).

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Figure 3. AHR is associated with a reduction in apparent affinity of 8-Br-cGMP for cGKI. A and B, Maxi-K currents in basilar artery cells from control (A) and AHR (B), recorded during test pulses to +80 mV, before and after addition of 500 μmol/L 8-Br-cGMP. C, Concentration-response relationship for activation of Maxi-K channel by 8-Br-cGMP; 3 to 10 cells at each concentration (44 cells from 21 CTR rats; 41 cells from 16 AHR); fit of the data to a standard logistic equation gave EC₅₀ values of 44 and 110 μmol/L, with Hill coefficients of 1.9; *P<0.05; **P<0.01.

Maxi-K Channel in Inside-Out Patches

Two general mechanisms could account for the 2.5-fold rightward shift in AHR, a reduction in affinity of 8-Br-cGMP for cGKI, or an alteration in the phosphoprotein target, the Maxi-K channel. To assess the latter, we studied the effect of exogenous cGKIα on Maxi-K channel activity in inside-out patches. Baseline values of open probability (membrane potential, 0 mV; 100 nmol/L free Ca²⁺, 1 mmol/L ATP) were 0.012±0.004 (9 patches/4 animals) versus 0.016±0.008 (11 patches/5 animals) in controls versus AHR, which were not different (by t test; P=0.61). After exposure of the cytoplasmic side to solution containing cGMP (100 μmol/L) and cGKIα (5 kU/mL), open probabilities increased by factors of 2.10±0.24 versus 2.46±0.19 in controls versus AHR, which were not different (by t test; P=0.26). These data indicated that activation of the Maxi-K channel by cGK was the same in controls and AHR, suggesting that the phosphorylation target of cGKI was not abnormal, and thus pointing to a possible reduction in affinity of 8-Br-cGMP for cGKI.

8-APT-cGMP

A reduction in apparent affinity of 8-Br-cGMP for cGKI can arise from a shift in proportion of cGKIα and cGKIβ, since these splice variants exhibit different relative sensitivities to different cGKI activators.¹¹ To assess the relative role of cGKIα versus cGKIβ in Maxi-K channel regulation, we studied the effect of 8-APT-cGMP, which exhibits a 200-fold greater selectivity for the α isoform compared with the β isoform.²⁴ In 7 cells from 3 control animals, 8-APT-cGMP (30 μmol/L) increased the Maxi-K current by 17±4%. This response was not changed by increasing the concentration to 100 μmol/L, indicating a saturating concentration of drug, and suggesting that this activator was less efficacious than 8-Br-cGMP (56±14% with 500 μmol/L). By comparison, the effect of 8-APT-cGMP (100 μmol/L) was significantly (by t test, P<0.05) reduced in AHR (6±5%; 5 cells from 2 animals). The decrease in apparent efficacy of 8-APT-cGMP in AHR suggested that an isoform sensitive to 8-Br-cGMP but not to 8-APT-cGMP, ie, cGKIβ, might be more active in AHR. To further investigate the relative roles of cGKIα versus cGKIβ, we perform experiments to measure protein and mRNA levels of these isomers.

cGKIα/β Versus cGKIβ Protein

We examined cGKIα and cGKIβ protein using two antibodies, one that is specific for the β isoform (cGKIβ antibody) and another that cannot discriminate between α and β isoforms (cGKIα/β antibody), since cGKIα is a shortened sequence of cGKIβ. For quantification, we used both deconvoluted immunofluorescence imaging and Western blots, with the former also being useful for localization. Tissue sections showed abundant labeling with cGKIα/β antibody in VSMC layers from controls (Figure 4A) that was clearly reduced in AHR (Figure 4B). Conversely, tissue sections showed labeling with cGKIβ antibody in VSMC layers from controls (Figure 4C) that was clearly increased in AHR (Figure 4D).

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Figure 4. Altered expression of α and β protein isoforms of cGKI in AHR. A through D, Deconvoluted immunofluorescence images of basilar artery sections labeled with antibody against cGKIα/β (A and B) or cGKIβ (C and D), for control (CTR) and AHR; bar=25 μm. E and F, Western blots and densitometric analyses of total protein (TP; 6 basilar arteries for both CTR and AHR), probed with antibody against cGKIα/β (E) or cGKIβ (F). G and H, Western blots and densitometric analyses of immunoprecipitated Slo protein (IP; 17 basilar arteries for both CTR and AHR), probed with antibody against cGKIα/β (G) or cGKIβ (H); *P<0.05; **P<0.01.

Western blots of control basilar artery total protein probed with the cGKIα/β antibody showed a prominent signal at ≈77 kDa, the expected molecular mass of monomeric cGKI (Figure 4E). Two distinct bands separated by <2 kDa were often appreciated, as previously reported.²⁵ This could not be attributed to the two isoforms, cGKIα (76 kDa) and cGKIβ (78 kDa), since in our hands recombinant cGKIα (Calbiochem, catalogue No. 370650) also exhibited two bands (not shown), but may have been related to proteolytic cleavage.²⁵ In homogenates of vessels from AHR, bands at 77 kDa were present but were less prominent than in controls (Figure 4E). Densitometric analysis confirmed that the signal for cGKIα/β was reduced to 70% of control values in AHR, a difference that was significant (by t test, P<0.01).

Western blots of basilar artery total protein from controls and AHR were also probed with the cGKIβ antibody. These blots showed a prominent band at ≈78 kDa that was appreciably stronger in AHR compared with controls (Figure 4F). Densitometry confirmed that the signal for cGKIβ was increased significantly (by t test, P<0.05), by a factor of 1.4 in AHR.

cGKIα/β Versus cGKIβ Associated With Maxi-K Channel

Maxi-K channels were immunoisolated to assess for association with cGKIα/β versus cGKIβ. Western blots of immunoisolated Maxi-K channels probed with the cGKIα/β antibody showed a strong band at ≈77 kDa (Figure 4G). In AHR, the signal was weaker compared with controls (Figure 4G), in agreement with previous observations made on total protein lysate. When blots of immunoisolates were probed using the cGKIβ antibody, the signal was visibly stronger in AHR compared with controls (Figure 4H), corroborating findings made with total protein lysates. In AHR, there was a 2-fold increase in cGKIβ associated with the Maxi-K channel, which was significant (by t test, P<0.01) (Figure 4H). Thus overall, there was less cGKI associated with Maxi-K channels in AHR, but the proportion representing the β isoform was significantly greater.

cGKIα Versus cGKIβ mRNA Transcription

We used real-time PCR to quantify mRNA for cGKIα and cGKIβ using probes unique for each. For cGKIα, levels of mRNA were reduced by two thirds in AHR compared with controls, which was significant (by t test, P<0.01) (Figure 5A). For cGKIβ, levels of mRNA were increased 18-fold in AHR compared with controls (t test, P<0.001). These findings corroborated the alterations in protein expression observed above.

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Figure 5. Altered transcription of α and β mRNA for cGKI in AHR. A, Bar graph showing fold change in mRNA for cGKIα and cGKIβ in AHR compared with control (5 rats each), with values of fold change in AHR indicated. B, Amplicons detected in real-time PCR experiments were confirmed as single products of the appropriate number of base pairs (187, 71, and 68, for 18S, cGKIα and cGKIβ, respectively). C, Northern blots for cGKIα and cGKIβ in control (CTR) and AHR (mRNA pooled from 5 basilar arteries each).

We used Northern blots to verify our measurements obtained with real-time PCR. Blots for both isoforms showed prominent signals in both controls and AHR, with mRNA for cGKIα showing a decrease in AHR, and mRNA for cGKIβ showing an increase in AHR (Figure 5C).

4d-Ang

Finally, we considered that alternative splicing resulting in a decrease in cGKIα mRNA and an increase in cGKIβ mRNA could result from either hypertension or from chronic Ang infusion. To further assess this, we performed quantitative immunofluorescence examination and Western blots of basilar arteries from rats exposed to Ang for only 4 days (4d-Ang), before development of hypertension. In these animals, basilar arteries showed labeling with the cGKIα/β antibody that was similar to controls (Figures 6A, 6B, and 6E). However, at 4 days, a strong increase in labeling for cGKIβ in VSMC layers was already evident in AHR (Figures 6C, 6D, and 6F). Quantification of the data indicated that the increase in β isoform at 4 days was significant (by ANOVA, P<0.01). Western blots of membrane versus cytoplasmic protein showed that at this early time, only cytosolic cGKI was elevated (Figures 6G and 6H). These data clearly implicated Ang and not hypertension per se as the cause for alternative splicing of cGKIα/β in AHR.

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Figure 6. Altered expression of cGKIβ protein develops before onset of hypertension. A through D, Deconvoluted immunofluorescence images of basilar artery sections labeled with antibody against cGKIα/β (A and B) or cGKIβ (C and D), for control (CTR) and 4d-Ang; bar=25 μm; control same as in Figure 4. E and F, Bar graphs of quantitative immunofluorescence imaging for cGKIα/β and cGKIβ, for control (CTR), 4d-Ang, and AHR (3 rats each); **P<0.01. G and H, Western blots and densitometric analyses of cytosolic (G) and membrane (H) protein (6 to 8 basilar arteries for both CTR and 4d-Ang), probed with antibody against cGKIβ; *P<0.05.