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. 2017 Jul 1;313(1):H207-H219.
doi: 10.1152/ajpheart.00480.2016. Epub 2017 May 26.

Chronic hypoxia attenuates the vasodilator efficacy of protein kinase G in fetal and adult ovine cerebral arteries

Richard B Thorpe  1 , Margaret C Hubbell  1 , Jinjutha Silpanisong  1 , James M Williams  1 , William J Pearce  2
Affiliations

Chronic hypoxia attenuates the vasodilator efficacy of protein kinase G in fetal and adult ovine cerebral arteries

Richard B Thorpe et al. Am J Physiol Heart Circ Physiol. .

Abstract

Long-term hypoxia (LTH) attenuates nitric oxide-induced vasorelaxation in ovine middle cerebral arteries. Because cGMP-dependent protein kinase (PKG) is an important mediator of NO signaling in vascular smooth muscle, we tested the hypothesis that LTH diminishes the ability of PKG to interact with target proteins and cause vasorelaxation. Prominent among proteins that regulate vascular tone is the large-conductance Ca2+-sensitive K+ (BK) channel, which is a substrate for PKG and is responsive to phosphorylation on multiple serine/threonine residues. Given the influence of these proteins, we also examined whether LTH attenuates PKG and BK channel protein abundances and PKG activity. Middle cerebral arteries were harvested from normoxic and hypoxic (altitude of 3,820 m for 110 days) fetal and adult sheep. These arteries were denuded and equilibrated with 95% O2-5% CO2 in the presence of N-nitro-l-arginine methyl ester (l-NAME) to inhibit potential confounding influences of events upstream from PKG. Expression and activity of PKG-I were not significantly affected by chronic hypoxia in either fetal or adult arteries. Pretreatment with the BK inhibitor iberiotoxin attenuated vasorelaxation induced by 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate in normoxic but not LTH arteries. The spatial proximities of PKG with BK channel α- and β1-proteins were examined using confocal microscopy, which revealed a strong dissociation of PKG with these proteins after LTH. These results support our hypothesis that hypoxia reduces the ability of PKG to attenuate vasoconstriction in part through suppression of the ability of PKG to associate with and thereby activate BK channels in arterial smooth muscle.NEW & NOTEWORTHY Using measurements of contractility, protein abundance, kinase activity, and confocal colocalization in fetal and adult ovine cerebral arteries, the present study demonstrates that long-term hypoxia diminishes the ability of cGMP-dependent protein kinase (PKG) to cause vasorelaxation through suppression of its colocalization and interaction with large-conductance Ca2+-sensitive K+ (BK) channel proteins in cerebrovascular smooth muscle. These experiments are among the first to demonstrate hypoxic changes in BK subunit abundances in fetal cerebral arteries and also introduce the use of advanced methods of confocal colocalization to study interaction between PKG and its targets.

Keywords: cGMP; chronic hypoxia; colocalization; confocal; guanylate cyclase; iberiotoxin.

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Figures

Fig. 1.
Fig. 1.
Effects of 8-pCPT-cGMP and hypoxia on 5-HT potency and concentration-response relations. A: pretreatment with graduated concentrations of 8-pCPT-cGMP determined the optimal concentration for investigating attenuation of vascular tone in ovine middle cerebral arteries. This approach demonstrated that hypoxia attenuated 5-HT-induced contraction in both fetal and adult arteries at the optimal concentration of 30 µM. B: adult hypoxic arteries exhibited modestly greater contractile responses and 5-HT potency compared with normoxic arteries. Increasing 8-pCPT-cGMP concentrations significantly attenuated 5-HT potency in all but the fetal hypoxic arteries. FN, fetal normoxic; FH, fetal hypoxic; AN, adult normoxic; AH, adult hypoxic. *P < 0.05. ns, Not significant.
Fig. 2.
Fig. 2.
Effects of 8-pCPT-cGMP and hypoxia on occupancy-response relations for 5-HT. A: 5-HT receptor affinity (pKa) was determined for each experimental group using the Furchgott method. The 5-HT concentration-response relations were converted to occupancy-response relations using pKa values to correct for differences in agonist binding affinity. *P < 0.05. B: the resulting occupancy-response relations revealed that compared with untreated control arteries, pretreatment with 30 µM 8-pCPT-cGMP significantly reduced maximum efficacy for 5-HT in normoxic (left) but not hypoxic (right) arteries from both age groups. *Significant differences (P < 0.05, repeated-measures ANOVA) between untreated and treated (30 µM 8-pCPT-cGMP) arteries. Error bars indicate means ± SE for n ≥ 5 for all groups.
Fig. 3.
Fig. 3.
PKG isoforms. A and B: abundance of total PKG was determined by Western blot analysis using an antibody against a PKG-I epitope common to both the Iα and Iβ isoforms. All abundances were calculated relative to known amounts of a standard pool prepared from normoxic adult arteries. Each gel included lanes with seven standards ranging from 1.0 to 15.0 µg and two samples for each of the four groups: normoxic fetal, hypoxic fetal, normoxic adult, and hypoxic adult. This approach evenly balanced the effects of gel-to-gel variation among all the experimental groups. B shows a group of contiguous unknown bands taken from a single gel-membrane image, for each antibody used. Relative abundance of PKG-Iβ was significantly (*P < 0.05) greater in fetal than adult arteries but was not affected by hypoxia. C: whole artery PKG activity in middle cerebral arteries (line graphs, pmol Pi/unit PKG) was not significantly affected by hypoxia in either fetal (left) or adult (right) homogenates. When whole artery PKG activity was normalized relative to PKG abundance (insets), estimates of specific activity (pmol Pi/min/unit PKG) did not vary significantly with hypoxia or between fetal and adult artery homogenates. These data indicate that the ability of hypoxia to ablate the effects of PKG on 5-HT contractions was not due to changes in PKG abundance or kinase activity. Error bars indicate means ± SE for n ≥ 6.
Fig. 4.
Fig. 4.
Concentration-response relations for 5-HT. A: effects of iberiotoxin, 8-pCPT-cGMP, age, and hypoxia on 5-HT concentration-response relations. Maximum contractile responses to graded concentrations of 5-HT varied moderately with age and hypoxia in untreated arteries. Maximum contractile responses in iberiotoxin-treated arteries were significantly greater in adult normoxic arteries compared with adult hypoxic arteries. Pretreatment with 30 µM 8-pCPT-cGMP significantly (*P < 0.05) attenuated 5-HT efficacy in a concentration-dependent manner in normoxic fetal and adult arteries. In arteries pretreated with 30 μM 8-pCPT-cGMP, 5-HT efficacy was greater in hypoxic compared with normoxic arteries for fetal but not adult arteries. Ctrl, control; IBX, iberiotoxin. B: vasorelaxation attributable to BK channel vs. non-BK channel vasorelaxation was determined using an algebraic model. Non-BK channel influence was of greater magnitude in all subgroups (FN, FH, AN, and AH), whereas BK channel effects were more sensitive to long-term hypoxia (LTH), particularly in fetal arteries. Error bars indicate means ± SE for n ≥ 6 for all groups. *Significance (P < 0.05).
Fig. 5.
Fig. 5.
BKα and BKβ1 isoforms. A: abundance of total BKα and BKβ1 isoforms was determined by Western blot analysis using an antibody against a specific epitope in both α- and β1-isoforms. B: all abundances were calculated relative to known amounts of a standard pool prepared from normoxic adult arteries. Relative abundance of BKα was significantly (*P < 0.05) attenuated (46%) by hypoxia in fetal cerebral arteries. Abundance of BKβ1 was not significantly influenced by chronic hypoxia. ANOVA showed a significant influence of hypoxia on fetal BKα abundance but no significant interactions of age with hypoxia for either BKα or BKβ1 protein abundances. Error bars indicate means ± SE for n ≥ 6.
Fig. 6.
Fig. 6.
Confocal microscopy. Top panels represent the distribution of BKα protein (red) and BKβ1 protein (green). The merged images appear as shades of yellow where the two proteins are colocalized within the resolution of the acquired images. Voxel dimensions were ≈146 × 146 × 545 nm at 488 nm (red) and ≈185 × 185 × 693 nm at 633 nm (green). Hypoxic artery segments in both fetal and adult arteries appeared visibly less yellow and thus represented less colocalization in that protein pair. Middle panels represent PKG (red) and BKα (green). These merged images likewise appeared visually to possess less colocalized protein for both fetal and adult hypoxic arteries. Bottom panels represent PKG (red) with BKβ1 (green) protein. Images represent a field of ~180 × 180 µm (scale, bottom right panel). Histological changes in the hypoxic arteries were also apparent; the medial layer was thinner and less invested in fusiform cell morphology, suggesting a probable shift in smooth muscle phenotype.
Fig. 7.
Fig. 7.
Quadrant analysis. A: representative scattergrams for fetal normoxic and hypoxic middle cerebral arteries illustrate a substantial loss of protein colocalization secondary to chronic hypoxia. Likewise, each of the six protein pairs exhibited qualitatively similar scattergrams (not shown). For the protein pair shown (BKα with BKβ1, fetal normoxic and hypoxic), the vertical axis (λ633, red) represents BKα and the horizontal axis (λ488, green) represents BKβ1 across the full resolution (intensities 0–255) of the analysis software. B: quadrant 1 (Q1; top right in each scattergram) colocalization expressed as a fraction of the total shows that chronic hypoxia had a significant effect on all three protein pairs (BKα with BKβ1, PKG with BKα, and PKG with BKβ1) in both fetal and adult arteries. Age (fetal vs. adult) displayed a modest but not significant trend in colocalization of BKα-with-BKβ1 and PKG-with-BKβ1 protein pairs in normoxic cerebral arteries (black bars). Age had no significant effect on colocalization of these protein pairs among hypoxic arteries (gray bars). *Significance (P < 0.05).
Fig. 8.
Fig. 8.
Protein proximity index (PPI). A: a three-dimensional mesh plot depicting the cross-correlation surface from an adult normoxic artery image stained for BKα and BKβ1. The horizontal scales represent a measure of Gaussian shift ±100 units from the peak intensity; the vertical scale represents signal intensity. The slow-decay portion of this surface (blues and yellows) represents a pseudocorrelation subset and was filtered from further analysis. The rapid-decay surface (oranges and reds) represents presumed protein-protein interactions (53). This cross-correlation surface, plus the autocorrelation surface for each individual protein of interest (not shown), provides a basis for computing the PPI. B: contour plot from the same data as above, showing the “slice” (straight black line) that defines a cross section for purposes of equation fitting of the grid data. The cross section always bisects the peak of each mesh plot and includes the rapid-decay surface. C: this PPI histogram shows that LTH significantly (*P < 0.05) depressed colocalization of BKα with BKβ1 as a ratio of total BKβ1 protein [(BKα + BKβ1)/BKβ1] in both fetal (F) and adult (A) arteries (1st panel). LTH also depressed the [(PKG + BKα)/PKG] and [(PKG+ BKβ1)/PKG] PPI in both fetal and adult arteries (3rd and 4th panels). LTH had less influence on the PPI of BKα colocalized with BKβ1 expressed as a ratio to total BKα [(BKα + BKβ1)/BKα] (5th panel). Age showed a small but significant (§P < 0.05) influence on the [(PKG + BKβ1)/BKβ1] PPI as well as the [(BKα + BKβ1)/BKα] and [(PKG + BKα)/BKα] indexes among hypoxic but not normoxic arteries (2nd, 5th, and 6th panels). Values of the PPI for [(PKG + RyR)/RyR] were significantly greater in normoxic fetal than normoxic adult arteries (§P < 0.05) and were significantly depressed by chronic hypoxia in fetal (*P < 0.05) but not adult arteries.
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