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. Author manuscript; available in PMC: 2016 Dec 17.
Published in final edited form as: Chem Biol. 2015 Dec 17;22(12):1653–1661. doi: 10.1016/j.chembiol.2015.11.005

Synthetic peptides as cGMP-independent activators of cGMP-dependent protein kinase Iα

Thomas M Moon a, Nathan R Tykocki a, Jessica L Sheehe a, Brent W Osborne b, Werner Tegge c, Joseph E Brayden a, Wolfgang R Dostmann a,
PMCID: PMC4703045  NIHMSID: NIHMS746603  PMID: 26687482

Abstract

PKG is a multifaceted signaling molecule and potential pharmaceutical target due to its role in smooth muscle function. A helix identified in the structure of the regulatory domain of PKG Iα suggests a novel architecture of the holoenzyme. In this study, a set of synthetic peptides (S-tides), derived from this helix was found to bind to and activate PKG Iα in a cGMP-independent manner. The most potent S-tide derivative (S1.5) increased the open probability (NPo) of the potassium channel KCa1.1 to levels equivalent to saturating cGMP. Introduction of S1.5 to smooth muscle cells in isolated, endothelium-denuded cerebral arteries through a modified reversible permeabilization (RP) procedure inhibited myogenic constriction. In contrast, in endothelium-intact vessels, S1.5 had no effect on myogenic tone. This suggests that PKG Iα activation by S1.5 in vascular smooth muscle would be sufficient to inhibit augmented arterial contractility that frequently occurs following endothelial damage associated with cardiovascular disease.

Introduction

In arterial smooth muscle, type I cGMP-dependent protein kinase (PKG I) functions as a central signaling node for the nitric oxide (NO) and natriuretic peptide pathways that activate soluble and particulate guanylyl cyclases, respectively (Bian and Murad, 2007; Hofmann et al., 2009; Kemp-Harper and Schmidt, 2009; Kuhn, 2003; Potter et al., 2009). The subsequent production of cyclic guanosine-3’,5’-monophosphate (cGMP) by these enzymes leads to the activation of PKG. This is accomplished through direct binding of the second messenger to the kinase (Lincoln et al., 1977; Pfeifer et al., 1999; Ruth et al., 1991). PKG I isoforms modulate excitation-contraction coupling in smooth muscle by reducing intracellular Ca2+ levels and promoting dephosphorylation of regulatory myosin light chains (Carrier et al., 1997; Feil et al., 2002; Kawada et al., 1997; Wellman et al., 1996; Wu et al., 1998). This is complemented by PKG Iα phosphorylating the large conductance Ca2+-activated potassium (KCa1.1/BK) channel (Schubert and Nelson, 2001). The PKG-mediated activation of the channel increases the flow of potassium ions across the membrane, hyperpolarizing the cell, inhibiting voltage dependent calcium channels (VDCCs), and thereby promoting vasodilation (Hofmann et al., 2014; Robertson et al., 1993; Sausbier et al., 2000).

All major therapies aimed at modulating blood flow through PKG-dependent mechanisms rely on regulating the rate of cGMP turnover, rather than the activity of PKG itself (Bryan et al., 2009; Evgenov et al., 2006; Kots et al., 2011; Salloum et al., 2012; Schlossmann and Hofmann, 2005). The use of PKG isoforms as pharmaceutical targets has been hampered by a lack of understanding of their mechanisms of activation due to their complex architectures. However, elucidating the regulatory mechanism of the PKG holoenzymes is central to guiding pharmacological discoveries relevant to prevention and treatment of vascular diseases. Recently, we solved the crystal structure of the intact regulatory domain of PKG I (Osborne et al., 2011). This structure exposed a unique helical domain that stabilizes an unexpected dimer interface between monomers in the asymmetric unit. Here we present the development of cGMP-independent peptide activators of PKG Iα, derived from this helical segment found bridging the regulatory and catalytic domains, and demonstrate their utility in intact tissue.

Results

PKG structure, design and synthesis of S-tides

The structure of of PKG Iα (78–355; PDB ID: 3SHR) identified an interaction formed by two bridging alpha helices between opposing protomers (Figure 1A) (Osborne et al., 2011). The complementary docking sites observed in either subunit are characterized by hydrophobic pockets located adjacent to the low-affinity cGMP binding sites. Each pocket buries over 1350 Å2 of surface area, and these interacting elements are conserved in PKG I isoforms (Moon et al., 2013). The C-terminal residues on the helix (F350, F351, N353, and L354) and a constellation of van der Waals and hydrogen bonds that form the aforementioned pocket were termed the “knob” and “nest,” respectively (Figure 1B). The hallmark of this interaction is the direct contact between the knob and residues that compose the nest, which also contribute to the formation of the phosphate binding cassette (PBC) (Figure 1C). It is the PBC — and its closure upon cyclic nucleotide binding — that facilitates the structural reorganization to release the catalytic domain in PKG. In full length PKG Iα, cooperativity was abolished through disruption of this unique interaction site (Osborne et al., 2011). Thus, we concluded that targeting the knob-nest site through synthetic, helical peptides may affect kinase activity. To this end, we synthesized the full alpha helical segment in PKG I comprising residues 329–358 (S1.1) and a series of peptides derived from this central sequence, subsequently denoted “S-tides” (Table 1).

Figure 1.

Figure 1

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Structure of the cGMP B-sites (B and B’) from the regulatory domain of PKG Iα (A) emphasizing the asymmetric interaction of helical segments between protomers (PDBID: 3SHR) (Osborne et al., 2011). The arrow indicates the directional view depicted in (B) detailing the interaction of the knob residues (F350’, F351’, L354’) with the nest (N353’ not depicted). C) Electrostatic surface representation of the cGMP B-site demonstrating its amino acid composition, the boundaries of the nest, and its location relative to the cGMP342 binding site.

Table 1.

S-tide composition and activation kinetics for PKG Iα

Name Sequence Ka (µM) nH N
S1.1 Ac-DVSNKAYEDAEAKAKYEAEAAFFANLKLSD-NH2 35 ±4 2.2 ±0.4 14
S1.2 Ac-DVSNKAYEDAEAKAKYEAEAAFFANLK-NH2 35 ±4 3.6 ±0.8 6
S1.3 Ac-DVSNKAYEDAEAKAKYEAEAA-NH2 - - 4
S1.4 Ac-NKAYEDAEAKAKYEAEAAFFANLKLSD-NH2 12 ±1 1.9 ±0.6 6
S1.5 Ac-YEDAEAKAKYEAEAAFFANLKLSD-NH2 3 ±1 2.4 ±0.6 8
S1.9 Ac-EDAEAKAKYEAEAAFFANLKLSD-NH2 43 ±5 2.7 ±0.7 6
S1.10 Ac-DAEAKAKYEAEAAFFANLKLSD-NH2 45 ±4 3.0 ±0.7 6
S1.11 Ac-AEAKAKYEAEAAFFANLKLSD-NH2 - 6
S1.6 Ac-DVSNKAYEDAEAKAKYEAEAAAAANLKLSD-NH2 - 4
S1.7 Ac-ALKSENYADKVEFKDAKYEASALANEFADA-NH2 - 4
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Preparation of cyclic nucleotide-free type I PKG isoforms

Previous purification methods of recombinant PKG I isoforms involved the use of cyclic nucleotide affinity chromatography, typically via agarose-linked cAMP (Dostmann et al., 2000; Feil et al., 1993). However, in order to examine the cyclic nucleotide-independent effects of the aforementioned peptides on PKG I activity, enzyme preparations were required that did not encounter elevated cyclic nucleotide concentrations during the purification process. To meet this need, we engineered PKG Iα and Iβ constructs carrying a cleavable N-terminal hexahistidine tag for isolation by Ni-IMAC chromatography (see methods).

For both isoforms, purified kinase preparations reconstituted in in vitro assays demonstrated similar kinetic constants as have been previously reported (Figure S1A, S1B) (Dostmann et al., 1999; Dostmann et al., 2000; Feil et al., 1995; Feil et al., 1993; Landgraf et al., 1991; Ruth et al., 1991; Scholten et al., 2007). We found that cleavage of the hexahistidine tag did not alter kinase activation kinetics (Figure S1C). To further examine the influence of the purification method on the activity of the enzyme, we isolated the His-tagged PKG Iα by 6-AEA-cAMP agarose affinity chromatography and observed no significant differences in its activation constants (Figure S1D). Although residual quantities of cAMP were detected following dialysis of PKG Iα purified by 6-AE-cAMP agarose, no detectable levels of cGMP or cAMP were found following the Ni-IMAC purification procedures of PKG Iα (Figure S2). In general, for our Ni-IMAC-purified PKG preparations, we observed low basal activities. This resulted in 20-fold and 15-fold increases in activity under saturating quantities of cGMP for PKG Iα and Iβ, respectively (Figure S1A, S1B).

S-tides act as cGMP-independent activators of PKG Iα

Any effect of S-tides on PKG Iα activity must be preceded by a binding event. Using surface plasmon resonance spectroscopy (SPR), we first examined the binding of the kinase to immobilized peptides. Repeated, single injections of PKG Iα on sensor chips covalently bound to a subset of S-tides found that PKG Iα associated with immobilized S1.1 and S1.5 (Figure S3Aa, S3Ab), but not S-tides lacking the knob residues by deletion or mutation (S1.3 and S1.6). The sequence-scrambled derivative (S1.7) also did not bind to the kinase (Figure S3Ac). While the projected maximal association was predicted to be within 10 minutes, this slow association was accompanied by an even slower dissociation of the kinase from the peptide-affixed surface (Figure S3Aa). To assess the reversibility of binding, peptide-affixed sensor chips were cleared of PKG using 1% SDS. Identical binding profiles were obtained for repeated injections of PKG followed by SDS washes. Next, PKG Iα was immobilized on the sensor chip and then probed with S-tides. Using this method, similar results were obtained (Figure S3B). However, these surfaces were unable to be regenerated by SDS, indicative of the sensitivity of PKG to denaturation by the detergent.

To examine the concentration-dependent effect of the S-tides on PKG activity, we needed to account for their apparent slow rate of association as assessed by SPR. Thus, all activity assays were performed under pre111 incubation conditions (15 minutes at 30°C, see methods). We observed robust and cooperative activation of PKG Iα by the parent compound, S1.1. As a control, we verified that S1.1 did not affect the KM for the substrate peptide W15 (Figure S4) (Dostmann et al., 1999). An activation constant of 35 µM in the absence of cGMP reached maximal efficacy of 80% as compared to cGMP controls (Figure 2A, Table 1). Shorter incubation periods resulted in dampening of the peptide’s potency (Ka = 300 µM) (Figure S5). Based on this observation, we concluded that our preincubation strategy provides the time necessary for S-tide association with the kinase, as corroborated by SPR.

Figure 2.

Figure 2

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Activation of PKG Iα by S-tides. Activity of PKG Iα was measured using peptides with (A) C-terminal truncations, modifications and (B) N-terminal truncation mutants derived from the parent compound S1.1. Activity is expressed as a percentage relative to 5µM cGMP for individual experiments. Values indicated are the mean +/− SD. Secondary structure determination of (C) C-terminal and (D) N-terminal truncation mutants as measured by CD spectroscopy. Mean residue ellipticity (θ) is expressed as mdeg × cm2/dmol × residue.

Next, a series of S1.1 derivatives was analyzed to probe the roles of the C-terminal amino acids in activating PKG Iα (Table 1). Deletion of the first three C-terminal residues (S1.2) maintained native potency (Ka=35 µM) but exhibited decreased efficacy (60% of maximum). Further deletion of C-terminal residues that include the knob (S1.3) completely abolished kinase activity (Figure 2A), suggesting that these residues play a vital role in promoting kinase activation. Finally, we found that alanine substitution of two knob residues (F350A and F351A, as found in peptide S1.6) was equivalent to removal of the entire knob. Likewise, a scrambled control of the parent peptide (S1.7) did not activate the kinase (Figure S5A).

In general, we found that this first set of S-tides possessed helical secondary structure as assessed by circular dichroism (CD) spectroscopy (Figure 2C). Peptides S1.1 and S1.2 displayed a similar degree of helicity; however, when the knob resides were deleted (S1.3), helicity was compromised but not completely lost. Mutation of the knob (S1.6) resulted in an increased degree of helicity. In contrast, the sequence-scrambled control (S.17) was found to not be helical (Figure S5B).

Next, we probed N-terminal deletions of S1.1. The S-tides S1.4 and S1.5, which had three and 6 amino acids truncated (Table 1), showed step-wise increases in potency to a minimal activation constant of 3 µM (S1.5), while retaining similar maximal velocities at 80% (Figures 2B and S6A). Further N-terminal truncations through single amino acid removal (S1.9–S.11) reversed this trend (Figures 2B and S6B). While the activation constants for S1.9 and S1.10 returned to values similar to S1.1, removal of one additional amino acid (D337, S1.11) resulted in a complete loss of activity. Moreover, for all N-terminal deletions, we observed a correlation between the loss in helicity and the loss in potency (Figures 2B, 2D, and S7). Given that S1.5 was found to be the most potent activator for PKG Iα, we investigated its efficacy and potency for activating PKG Iβ. Interestingly, we observed no detectable activation of PKG Iβ using the S1.5 peptide (Figure S8).

S1.1 and S1.5 but not scrambled peptide increases KCa1.1 channel open probability in inside-out patches from VSM

To assess the functional relevance of the S-tides, we investigated their effects on the large conductance calcium-activated potassium channel (KCa1.1), as it is one of the few well-established molecular targets of PKG Iα in VSM (Robertson et al., 1993; Sausbier et al., 2000). Inside-out membrane patches derived from freshly isolated cerebral artery myocytes demonstrated consistent KCa1.1 channel openings in the presence of 500 nM Ca2+ for every experimental condition tested (Figure 3A, left panel). Upon addition of recombinant PKG Iα to the bath solution (cytosolic face) and in the presence of 50 nM cGMP to mimic basal levels of cGMP typically found in VSM (Francis et al., 1988; Jiang et al., 1992), we observed no significant change in KCa1.1 open probability (NPo) (Figure 3A, right panel, 3B). The addition of 5 µM cGMP to evoke saturating conditions was sufficient to raise the NPo eight-fold above the control (p<0.002) (Figure 3B). Similar to the activation by saturating cGMP, we observed that 100 µM S1.1 pre-incubated with PKG Iα substantially increased (6-fold, p<0.05) KCa1.1 channel activity. Likewise, 10 µM S1.5 gave near-identical results (5-fold, p<0.002). This increase in NPo is consistent with PKG-mediated stimulation of the channel as previously described (Alioua et al., 1995; Alioua et al., 1998). In contrast, introduction of the scrambled control peptide S1.7 did not stimulate KCa1.1 (Figure 3A, lower panel).

Figure 3.

Figure 3

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Effects of peptide- or cGMP-treated PKG Iα added to the cytosolic face of excised, inside-out membrane patches containing KCa1.1 channels A) Representative control (left) and experimental traces (right) of single KCa1.1 channel openings in excised membrane patches from cerebral artery myocytes. The combined results of single KCa1.1 recordings (B) demonstrate a substantially increased NPo versus control for saturating cGMP levels and S-tides S1.1 and S1.5 († P<0.002, * P<0.05). Mean values with SEM are represented. Dashed line indicates normalized channel activity from control patches.

Paxilline-induced constriction is augmented in S1.5-treated arteries

Smooth muscle KCa1.1 activation represents a predominant vasodilatory, negative feedback mechanism in myogenically-active blood vessels (Nelson et al., 1995). Thus, we investigated the physiological effects of S1.5 on vessel diameter in the presence or absence of the KCa1.1 blocker paxilline. We selected S1.5 for its potency and selectivity toward recombinant PKG Iα. Because S1.5 was unable to cross the plasma membrane unaided, we introduced the molecule to intact arteries using a reversible permeabilization (RP) procedure as previously described (Earley et al., 2004) (see methods). RP utilizes the activation of P2X7 receptors that are specifically expressed in endothelium and VSM cells (Lesh et al., 1995).

To test whether S1.5 was able to enter VSM cells through RP, we synthesized an S1.5 analog containing an N-terminal fluorescein tag (FITC-S1.5). Posterior cerebral arteries (PCA) were exposed to either S1.5 or FITC-S1.5 using the RP protocol. Individual VSM cells were dissociated using papain and collagenase, and analyzed by confocal fluorescence microscopy. Smooth muscle cells from arteries treated with FITC-S1.5 showed clear internalization of the peptide with diffuse staining throughout the cytosol. VSM cells from arteries treated with non-labeled S1.5 did not fluoresce (Figure 4).

Figure 4.

Figure 4

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Posterior cerebral arteries were subjected to reverse permeabilization with FITC-βAla2-S1.5 (experimental, left panel) and S1.5 (control, right panel) peptides. Following digestion using papain and collagenase, single smooth muscle cells were imaged using differential interference contrast microscopy (DIC) and confocal fluorescence microscopy (CFM).

Next, we studied the functional effects of S1.5 in isolated arteries. S1.5 significantly reduced myogenic tone development in endothelium-denuded PCAs, as compared to controls (39% vs. 66%; p<0.05) (Figure 5Aa, 5Ab, 5Ac). Subsequent addition of the KCa1.1 inhibitor paxilline (1 µM) resulted in an additional constriction that was significantly greater in S1.5-treated arteries than controls (26% vs. 5%; p<0.001) (Figure 5Ad). Interestingly, the maximal constriction (% constriction in the presence of paxilline) was not different between control and S1.5 treated arteries (Figure 5Ae). This indicates that the reduction in myogenic tone in the S1.5-treated arteries was due primarily to augmented KCa1.1 activity.

Figure 5.

Figure 5

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A) Diameter measurements for endothelium-denuded, reversibly permeabilized arteries demonstrating myogenic tone response in (Aa) control and (Ab) S1.5-treated arteries. (Ac-Ae) Summarized results of diameter measurements for endothelium-denuded arteries in control and permeabilized treatments are shown with values represented as the mean +/− SEM. Differences in myogenic tone in response to pressure (Ac) as well as paxilline-induced constriction (Ad) are shown (P<0.05). Diameter measurements from endothelium-intact arteries for controls (Ba) and S1.5-treated arteries (Bb) show unaltered myogenic tone response (Bc) but small differences in paxilline-induced constriction of control and S1.5 treated arteries (Bd, P<0.02). For both sets of experiments, total constriction (Ae, Be) was unchanged between controls and S1.5-treated arteries.

In PCAs with intact endothelium, we observed no significant difference in myogenic tone between control and S1.5-treated arteries, but found that paxilline caused a larger constriction in S1.5 treated arteries as compared to controls (12% vs. 4%; p<0.02) (Figure 5Ba–d). However, as with endothelium-denuded vessels, maximal constriction in the presence of paxilline was not different between control and S1.5 treated arteries (Figure 5Be). These data indicate that activation of endogenous PKG Iα by S1.5 leads to a net, KCa1.1 channel-driven relaxation of smooth muscle cells in intact posterior cerebral arteries.

Discussion

PKG I activation is mediated through the modulation of cellular levels of cGMP and the activity of this kinase is a critical component of vascular function (Francis et al., 2010; Hofmann et al., 2009). The cGMP-independent modulation of PKG Iα activity in relation to KCa1.1 activity represents an attractive avenue toward novel therapeutic treatments of age-related cardiovascular disease. Traditionally, exogenous stimulation of PKG has been accomplished by the use of cGMP analogs (Butt, 2009; Schwede et al., 2000). Although these compounds have served as valuable tools for studying PKG signaling, their clinical utility has remained elusive due to off-target effects, such as cross-activation of other cyclic nucleotide-binding proteins and modulation of phosphodiesterase activity. Here, we introduce a set of synthetic peptide activators (S-tides) derived from a novel structural element within the PKG I protein kinase. S-tides represent a potential new class of compounds, capable of eliciting vasodilation through a mechanism similar to that of nitrates and phosphodiesterase inhibitors (i.e., activation of PKG), but without the need for cGMP (Bryan et al., 2009; Kots et al., 2011; Schlossmann and Hofmann, 2005).

Biochemistry

We observed that isoform-specific PKG Iα activity can be promoted by S-tides in a cGMP-independent manner. Truncation of the C-terminus of the S1.1 parent peptide in a form that removes or modifies the knob residues (F350, F351) produces peptides that do not stimulate kinase activity. The N-terminal truncations of the parent molecule provided an increase in potency followed by a dramatic reduction and, u ltimately, a complete loss of activity that was dependent on how many amino acids were removed. Concomitant with these shifts in the activation constant, we observed a positive correlation between activation of the kinase and helicity (Figure S7). We propose that stabilization of this helix by chemical means, as has been demonstrated with AKAP disruptors, may pose an additional avenue for further development of these activators (Kennedy and Scott, 2015; Wang et al., 2015).

The most potent peptide activator for PKG Iα (S.15) was unable to activate PKG Iβ, even though the type I isoforms of PKG retain 99% identity outside of their dimerization and autoinhibitory domains (Hofmann, 1995; Orstavik et al., 1997; Pfeifer et al., 1999; Sandberg et al., 1989; Wernet et al., 1989). The 10-fold difference in activation constants observed for PKG I isoforms by cGMP (Figure S1A, S1B) has been linked directly to these regions in the N-terminus (Ruth et al., 1997). The selectivity of S-tides for PKG Iα over Iβ may be governed by these differences.

The cGMP B-site can be divided into two parts; one containing the nest, and the other containing the cGMP-binding site (Figure 1C). Of note, part of the nest is formed by residues that construct the cGMP binding site. Residue W288 in addition to E291 and L294 from the phosphate binding cassette (PBC) help form the ridge of the nest (Figure 1B). In the reported structure, L294 comes into proximity with F351’ upon docking of the knob. It is feasible that the interaction of the knob with the nest may affect the geometry of the PBC to mimic a closed, cGMP-bound state, not captured in the reported structure. Even in the absence of supporting structural data, we found that deletion or mutation of the knob residues in S-tides resulted in retarded activation of the kinase. This suggests a mechanism indicative of the knob/nest interaction observed in the crystal structure of the intact regulatory domain. In any case, these molecules are efficacious and provide an unexplored avenue for the development of potent and selective cGMP-independent activators of PKG Iα.

Vascular Biology

In inside-out patches from posterior cerebral artery myocytes, S-tide incubation with PKG Iα stimulated KCa1.1 activity to a similar magnitude as saturating levels of cGMP. Basal level activity of PKG Iα in the presence of low nanomolar quantities of cGMP did not affect KCa1.1 open probability. Based upon these findings, we advanced the hypothesis that S-tide mediated stimulation of PKG Iα provides a basis for future attempts to modify PKG activity and effect physiological responses. This hypothesis was further supported by our observation that cerebral vascular myogenic tone was reduced in endothelium-denuded arteries exposed to S1.5. Of particular interest was our observation that paxilline-induced constriction was significantly augmented, suggesting that S1.5 stimulates KCa1.1 activity in vascular myocytes in situ. The minimal augmentation of tone by paxilline in endothelium-intact vessels treated with S1.5 suggests that simultaneous activation of endothelial PKG and smooth muscle PKG has opposing effects. There is precedent for this interpretation, as activation of endothelial PKG acts as a negative regulator of NO release (Borysova and Burdyga, 2015; Dora et al., 2001). Our finding that endothelium-denuded vessels have decreased myogenic tone in the presence of S1.5 further reinforces this concept, and shows that activation of smooth muscle PKG could be sufficient to oppose the exaggerated myogenic tone that develops in response to endothelial damage associated with cardiovascular diseases such as hypertension and diabetes (Sun et al., 1994)(Huang et al., 1993; Zimmermann et al., 1997). An important advantage of this type of potential therapeutic intervention is that S1.5 does not appear to negatively affect the development of tone in vessels where the endothelium is functioning normally.

Significance Statement

The control of vascular smooth muscle dilation and the regulation of blood flow are tightly linked to the rise and fall of cGMP, a small molecule that is made by vascular cells and is integral to proper circulatory function. This molecule controls the activation of a key enzyme, the cGMP-dependent protein kinase (PKG Iα), which is responsible for controlling a host of processes that regulate dilation of blood vessels independent of input from other body systems. All existing therapeutics that target this pathway either increase cGMP production or inhibit its breakdown. Here, we describe a novel class of small molecules, called S-tides, capable of activating PKG Iα, directly and selectively. S-tides also blunted the excessive constriction of blood vessels typical of arteries whose innermost layer is destroyed, which is often seen in hypertension and diabetes.

Materials and Methods

Recombinant Expression and Purification of PKG I isoforms

Expression in Sf9 cells using the Bac-to-Bac Baculovirus system (Life Technologies) was as previously reported (Dostmann et al., 2000). His-tagged proteins were isolated from Sf9 cells by homogenization followed by trapping on a Mini Profinity IMAC column (BioRad). Proteins were eluted using 250 mM imidazole and dialyzed into a final buffer of 50 mM MES, pH=6.9, 100 mM NaCl, 1 mM TCEP, and 10% glycerol. Full methods can be found in the SI Materials and Methods.

Peptide Synthesis

The solid-phase synthesis of the peptides was carried out as previously described (Nickl et al., 2010; Tegge et al., 2010). A full description of the methods can be found in the SI Materials and Methods.

Surface Plasmon Resonance

SPR measurements were conducted on a SR7500 dual-channel surface plasmon resonance spectrometer connected to an SR7100 autosampler using a standard, dual channel flow cell (Reichert Technologies). Gold sensorchips coated with 500 kDa carboxymethyl dextran hydrogel (Reichert Technologies) were used to immobilize PKG Iα or S-tides to the surface using EDC/NHS coupling chemistry at 25°C (Schasfoort and Tudos, 2008). Typically, 250nM PKG Iα was injected onto the surface for 7 minutes. Dissociation times of 15 minutes were recorded. A full description can be found in the SI Materials and Methods

Kinetic Analysis

Activity of recombinant PKG Iα toward the synthetic substrate peptide (W15, TQAKRKKSLAMA) was measured by γ32P-ATP incorporation assay as described with some modifications (Dostmann et al., 2000). Briefly, reactions were initiated when 0.1 mM γ32P-ATP (200–300 cpm/pmol) was incubated with vials preincubated with 50 mM MES, pH=6.9, 1 mM MgAcetate, 10 mM NaCl, 10 mM DTT, 1 mg/mL BSA, 10 µM W15 substrate, 1 nM PKG Iα, and 10 µL of 10× S-tide stocks, in 100 µL reaction volume at 30°C. Prior to the initiation of the reaction, all components excluding γ32P-ATP were allowed to pre-incubate for 15 minutes. Each reaction was run for 90 seconds and terminated by blotting on 25 mm phosphocellulose circles (Whatman P81 filter paper, GE Life Sciences). Filters were washed 3 times in 0.8% phosphoric acid and measured by liquid scintillation counting. Data was analyzed using Excel (Microsoft), Prism 6 (GraphPad), and plotted using DataGraph (Visual Data Tools).

Circular Dichroism Spectroscopy

CD spectra were collected on a JASCO J-815 circular dichroism spectrometer at 22°C using a 2mm cuvette (Starna Cells). Data were collected on samples containing 10 µM peptide in 10 mM PBS pH=7.4. A sampling range from 260 to 190 nm with a 0.5 nm sampling interval and 2 s integration time. A total of 10 accumulations were collected at 100 nm/min and averaged to produce the final spectra for each peptide.

Electrophysiology

Potassium channel activity was recorded as previously described (Brayden and Nelson, 1992). Briefly, inside284 out membrane patches were obtained from enzymatically dispersed cerebral artery smooth muscle cells. The bathing solution (intracellular face) contained (in mmol/L) 140 KCl, 2 MgCl2, 0.1 Mg-ATP, 10 HEPES (pH=7.3), and 3 EGTA. CaCl2 was added to the bath solution to achieve a concentration of 500 nM. The pipette solution contained (in mmol/L) 120 NaCl, 20 KCl, 2 MgCl2, 1 CaCl2, and 10 HEPES (pH=7.4). Single BK channel recordings were obtained over at least 5 minutes at a holding potential of +10mV in the absence and presence of purified PKG and various derivatives of the SW peptide. See SI Materials and Methods for a complete description.

Reversible Permeabilization and Myography

To introduce S1.5 peptide into the smooth muscle cytosol, isolated rat posterior cerebral arteries were reversibly permeabilized as described previously, see SI Materials and Methods (Earley et al., 2004; Lesh et al., 1995). Vessels were then placed in physiological saline solution (PSS) containing (in mM): 118.5 NaCl, 4.7 KCl, 1.2 KH2PO4, 24 NaHCO3, 1.2 MgCl2, 2.0 CaCl2, 11 glucose, 0.026 EDTA (pH 7.4 at 37°C), and cannulated to resistance-matched micropipettes filled with PSS. Endothelial denudation was achieved by passing air bubbles through the vessel lumen. Intraluminal pressure was increased to 40 or 80 mmHg using an electronic servo-pressure transducer (Living Systems, St. Albans, VT USA), and myogenic tone was allowed to develop (~45 minutes) prior to the addition of drugs. In endothelium-denuded vessels, denudation was confirmed by the absence of vasodilation to the SK/IK activator NS-309 (1 µM). During the experiment, vessel diameter was monitored continuously using a CCD camera and edge-detection software (Living Systems). At the end of each experiment, vessels were exposed to Ca2+-free PSS containing 5 mM EGTA to determine the maximal passive diameter. All data were then normalized to the maximal passive diameter.

Smooth Muscle Cell Isolation and Confocal Fluorescence

Microscopy S1.5 peptide or FITC-S1.5 peptide were introduced into rat posterior cerebral arteries as described above. Vessels were then digested, using papain (0.5 mg/ml) with dithioerythritol (1 mg/ml) followed by Type F collagenase (1 mg/ml; Sigma Aldrich, USA). Smooth muscle cells were isolated by gentle trituration, and allowed to adhere to glass coverslips at RT for 30 minutes. Cells were then imaged using a Zeiss LSM 510 Meta confocal laser scanning microscope with a 63× oil-immersion objective (NA=1.4). Identical imaging conditions were used for smooth muscle cells containing control (S1.5) and fluorescent (FITC-S1.5) peptide.

Statistical Analysis

Arterial diameter data were analyzed using LabChart 7 Pro software (ADInstruments, Colorado Springs, CO, USA). Electrophysiological data were analyzed using pCLAMP 9 software (Molecular Devices). For comparisons of two samples of equal variance, statistical significance between groups was determined using two-tailed, unpaired Student’s t-tests (α = 0.05). For multiple sample comparisons, one- and two-way ANOVA were used followed by Bonferroni’s post hoc analysis to compare individual means. P-values ≤ 0.05 were considered significant. These and all other relevant calculations were performed using Microsoft Excel (Microsoft Corporation, USA) and GraphPad Prism (GraphPad Software Inc., USA).

Highlights.

  • S-tides are a new class of molecule capable of selectively activating PKG Iα

  • S-tides target PKG Iα and elevate the open probability of K+ channels (BK, KCa1.1).

  • S-tides modulate vascular contractility via PKG Iα effects on BK channels.

  • S-tides dilate damaged arteries that have exaggerated myogenic tone.

Acknowledgements

We would like to thank Drs. Frank Schwede at BioLog, Bremen, Germany for HPLC analyses of cyclic nucleotide samples and Matthew Liptak at the University of Vermont Department of Chemistry for use of the CD spectrometer. This research was supported by grants from the National Institutes of Health (NIH) with direct support to TMM (5T32 HL007647) and NRT (1K01 DK103840), JEB (P01 HL095488), and WRD (R01 HL68891) as well as support from the Totman Trust for Biomedical Research. A U.S. Non-provisional patent application 13/801,235, was filed March 13, 2013, entitled "NOVEL PEPTIDIC ACTIVATORS OF TYPE I cGMP DEPENDENT PROTEIN KINASES AND USES THEREOF" by Wolfgang Dostmann, Brent W. Osborne, and Thomas M. Moon. We would like to dedicate this manuscript to the memory of Matthew J. Tavares.

Footnotes

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Author Contributions

T.M.M., W.R.D., N.R.T., and J.E.B. conceived of and designed the experiments. T.M.M., N.R.T, W.T., J.S., and B.W.O. performed experiments. All authors analyzed the data. T.M.M. and W.R.D. wrote the manuscript with contributions from N.R.T. and J.E.B.

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