Pressure-dependent contribution of Rho kinase-mediated calcium sensitization in serotonin-evoked vasoconstriction of rat cerebral arteries.

Our understanding of the cellular signalling mechanisms contributing to agonist-induced constriction is almost exclusively based on the study of conduit arteries. Resistance arteries/arterioles have received less attention as standard biochemical approaches lack the necessary sensitivity to permit quantification of phosphoprotein levels in these small vessels. Here, we have employed a novel, highly sensitive Western blotting method to assess: (1) the contribution of Ca(2+) sensitization mediated by phosphorylation of myosin light chain phosphatase targeting subunit 1 (MYPT1) and the 17 kDa PKC-potentiated protein phosphatase 1 inhibitor protein (CPI-17) to serotonin (5-HT)-induced constriction of rat middle cerebral arteries, and (2) whether there is any interplay between pressure-induced myogenic and agonist-induced mechanisms of vasoconstriction. Arterial diameter and levels of MYPT1 (T697 and T855), CPI-17 and 20 kDa myosin light chain subunit (LC(20)) phosphorylation were determined following treatment with 5-HT (1 micromol l(1)) at 10 or 60 mmHg in the absence and presence of H1152 or GF109203X to suppress the activity of Rho-associated kinase (ROK) and protein kinase C (PKC), respectively. Although H1152 and GF109203X suppressed 5-HT-induced constriction and reduced phospho-LC(20) content at 10 mmHg, we failed to detect any increase in MYPT1 or CPI-17 phosphorylation. In contrast, an increase in MYPT1-T697 and MYPT1-T855 phosphorylation, but not phospho-CPI-17 content, was apparent at 60 mmHg following exposure to 5-HT, and the phosphorylation of both MYPT1 sites was sensitive to H1152 inhibition of ROK. The involvement of MYPT1 phosphorylation in the response to 5-HT at 60 mmHg was not dependent on force generation per se, as inhibition of cross-bridge cycling with blebbistatin (10 micromol l(1)) did not affect phosphoprotein content. Taken together, the data indicate that Ca(2+) sensitization owing to ROK-mediated phosphorylation of MYPT1 contributes to 5-HT-evoked vasoconstriction only in the presence of pressure-induced myogenic activation. These findings provide novel evidence of an interplay between myogenic- and agonist-induced vasoconstriction in cerebral resistance arteries.

ADP signaling in vascular endothelial cells: ADP-dependent activation of the endothelial isoform of nitric-oxide synthase requires the expression but not the kinase activity of AMP-activated protein kinase.

ADP responses underlie therapeutic approaches to many cardiovascular diseases, and ADP receptor antagonists are in widespread clinical use. The role of ADP in platelet biology has been extensively studied, yet ADP signaling pathways in endothelial cells remain incompletely understood. We found that ADP promoted phosphorylation of the endothelial isoform of nitric-oxide synthase (eNOS) at Ser(1179) and Ser(635) and dephosphorylation at Ser(116) in cultured endothelial cells. Although eNOS activity was stimulated by both ADP and ATP, only ADP signaling was significantly inhibited by the P2Y(1) receptor antagonist MRS 2179 or by knockdown of P2Y(1) using small interfering RNA (siRNA). ADP activated the small GTPase Rac1 and promoted endothelial cell migration. siRNA-mediated knockdown of Rac1 blocked ADP-dependent eNOS Ser(1179) and Ser(635) phosphorylation, as well as eNOS activation. We analyzed pathways known to regulate eNOS, including phosphoinositide 3-kinase/Akt, ERK1/2, Src, and calcium/calmodulin-dependent kinase kinase-beta (CaMKKbeta) using the inhibitors wortmannin, PD98059, PP2, and STO-609, respectively. None of these inhibitors altered ADP-modulated eNOS phosphorylation. In contrast, siRNA-mediated knockdown of AMP-activated protein kinase (AMPK) inhibited ADP-dependent eNOS Ser(635) phosphorylation and eNOS activity but did not affect eNOS Ser(1179) phosphorylation. Importantly, the AMPK enzyme inhibitor compound C had no effect on ADP-stimulated eNOS activity, despite completely blocking AMPK activity. CaMKKbeta knockdown suppressed ADP-stimulated eNOS activity, yet inhibition of CaMKKbeta kinase activity using STO-609 failed to affect eNOS activation by ADP. These data suggest that the expression, but not the kinase activity, of AMPK and CaMKKbeta is necessary for ADP signaling to eNOS.

Identification and functional characterization of protein kinase A-catalyzed phosphorylation of potassium channel Kv1.2 at serine 449.

Vascular smooth muscle Kv1 delayed rectifier K+ channels (KDR) containing Kv1.2 control membrane potential and thereby regulate contractility. Vasodilatory agonists acting via protein kinase A (PKA) enhance vascule smooth muscle Kv1 activity, but the molecular basis of this regulation is uncertain. We characterized the role of a C-terminal phosphorylation site, Ser-449, in Kv1.2 expressed in HEK 293 cells by biochemical and electrophysiological methods. We found that 1) in vitro phosphorylation of Kv1.2 occurred exclusively at serine residues, 2) one major phosphopeptide that co-migrated with 449pSASTISK was generated by proteolysis of in vitro phosphorylated Kv1.2, 3) the peptide 445KKSRSASTISK exhibited stoichiometric phosphorylation by PKA in vitro, 4) matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectroscopy (MS) and MS/MS confirmed in vitro Ser-449 phosphorylation by PKA, 5) in situ phosphorylation at Ser-449 was detected in HEK 293 cells by MALDI-TOF MS followed by MS/MS. MIDAS (multiple reaction monitoring-initiated detection and sequencing) analysis revealed additional phosphorylated residues, Ser-440 and Ser-441, 6) in vitro 32P incorporation was significantly reduced in Kv1.2-S449A, Kv1.2-S449D, and Kv1.2-S440A/S441A/S449A mutant channels, but Kv1.2-S440A/S441A was identical to wild-type Kv1.2 (Kv1.2-WT), and 7) bath applied 8-Br-cAMP or dialysis with PKA catalytic subunit (cPKA) increased Kv1.2-WT but not Kv1.2-S449A current amplitude. cPKA increased Kv1.2-WT current in inside-out patches. Rp-CPT-cAMPS reduced Kv1.2-WT current, blocked the increase due to 8-Br-cAMP, but had no effect on Kv1.2-S449A. cPKA increased current due to double mutant Kv1.2-S440A/S441A but had no effect on Kv1.2-S449D or Kv1.2-S440A/S441A/S449A. We conclude that Ser-449 in Kv1.2 is a site of PKA phosphorylation and a potential molecular mechanism for Kv1-containing KDR channel modulation by agonists via PKA activation.

Ca2+ sensitization via phosphorylation of myosin phosphatase targeting subunit at threonine-855 by Rho kinase contributes to the arterial myogenic response.

Ca(2+) sensitization has been postulated to contribute to the myogenic contraction of resistance arteries evoked by elevation of transmural pressure. However, the biochemical evidence of pressure-induced increases in phosphorylated myosin light chain phosphatase (MLCP) targeting subunit 1 (MYPT1) and/or 17 kDa protein kinase C (PKC)-potentiated protein phosphatase 1 inhibitor protein (CPI-17) required to sustain this view is not currently available. Here, we determined whether Ca(2+) sensitization pathways involving Rho kinase (ROK)- and PKC-dependent phosphorylation of MYPT1 and CPI-17, respectively, contribute to the myogenic response of rat middle cerebral arteries. ROK inhibitors (Y27632, 0.03-10 micromol l(-1); H1152, 0.001-0.3 micromol l(-1)) and PKC inhibitors (GF109203X, 3 micromol l(-1); Gö6976; 10 micromol l(-1)) suppressed myogenic vasoconstriction between 40 and 120 mmHg. An improved, highly sensitive 3-step Western blot method was developed for detection and quantification of MYPT1 and CPI-17 phosphorylation. Increasing pressure from 10 to 60 or 100 mmHg significantly increased phosphorylation of MYPT1 at threonine-855 (T855) and myosin light chain (LC(20)). Phosphorylation of MYPT1 at threonine-697 (T697) and CPI-17 were not affected by pressure. Pressure-evoked elevations in MYPT1-T855 and LC(20) phosphorylation were reduced by H1152, but MYPT1-T697 phosphorylation was unaffected. Inhibition of PKC with GF109203X did not affect MYPT1 or LC(20) phosphorylation at 100 mmHg. Our findings provide the first direct, biochemical evidence that a Ca(2+) sensitization pathway involving ROK-dependent phosphorylation of MYPT1 at T855 (but not T697) and subsequent augmentation of LC(20) phosphorylation contributes to myogenic control of arterial diameter in the cerebral vasculature. In contrast, suppression of the myogenic response by PKC inhibitors cannot be attributed to block of Ca(2+) sensitization mediated by CPI-17 or MYPT1 phosphorylation.

Amyloid beta peptides mediate physiological remodelling of the acute O2 sensitivity of adrenomedullary chromaffin cells following chronic hypoxia.

OBJECTIVE: The non-neurogenic response of the neonatal adrenal medulla is vital in cardiovascular and respiratory development and to the survival of newborns exposed to hypoxic stress. Here, we examined the acute hypoxic response of immortalised rat adrenomedullary chromaffin cells following exposure to chronic hypoxia (CH; 6% O(2) for 24 h). METHODS: Ca(2+) and K(+) channel currents were recorded using by whole-cell patch-clamp. RESULTS: Following incubation in CH, the acute O(2) sensitivity of K(+) current in immortalised adrenomedullary chromaffin (MAH) cells was enhanced due to a selective increase in the density of an O(2)-sensitive Ca(2+)-dependent K(+) current, secondary to ROS-mediated augmentation of voltage-gated Ca(2+) currents. The effect of CH on Ca(2+) currents was not additive to exogenous Abeta(1-40) and was blocked by the gamma-secretase inhibitors gamma-X and gamma-VI, demonstrating a role for amyloid beta peptide (AbetaP) production. Ca(2+) current enhancement was abolished in the presence of the transcription inhibitor actinomycin D but unaffected by the vacuolar H(+) ATPase inhibitor bafilomycin A1. CONCLUSION: AbetaP production and transcriptional regulation during CH regulated the properties of a peripheral chemosensory cell, defining a role for these enigmatic peptides in the signalling pathway of a physiological response to CH in the developing cardiovascular system.

System-specific O2 sensitivity of the tandem pore domain K+ channel TASK-1.

Hypoxic inhibition of TASK-1, a tandem pore domain background K+ channel, provides a critical link between reduced O2 levels and physiological responses in various cell types. Here, we examined the expression and O2 sensitivity of TASK-1 in immortalized adrenomedullary chromaffin (MAH) cells. In physiological (asymmetrical) K+ solutions, 3 microM anandamide or 300 microM Zn2+ inhibited a strongly pH-sensitive current. Under symmetrical K+ conditions, the anandamide- and Zn2+-sensitive K+ currents were voltage independent. These data demonstrate the functional expression of TASK-1, and cellular expression of this channel was confirmed by RT-PCR and Western blotting. At concentrations that selectively inhibit TASK-1, anandamide and Zn2+ were without effect on the magnitude of the O2-sensitive current or the hypoxic depolarization. Thus TASK-1 does not contribute to O2 sensing in MAH cells, demonstrating the failure of a known O2-sensitive K+ channel to respond to hypoxia in an O2-sensing cell. These data demonstrate that, ultimately, the sensitivity of a particular K+ channel to hypoxia is determined by the cell, and we propose that this is achieved by coupling distinct hypoxia signaling systems to individual channels. Importantly, these data also reiterate the indirect O2 sensitivity of TASK-1, which appears to require the presence of an intracellular mediator.

GABA(B) receptor activation augments TASK-1 in MAH cells and mediates autoreceptor feedback during hypoxia.

Previously, we demonstrated an autoregulatory feedback loop in the rat carotid body (CB), involving presynaptic GABA(B) receptor-mediated activation of the background K(+) channel TASK-1. Here, we examined the effects of the selective GABA(B) receptor agonist baclofen on K(+) currents in immortalised adrenomedullary chromaffin (MAH) cells, which share the same sympathoadrenal lineage as CB type I cells. Under symmetrical K(+) conditions, 50 microM baclofen enhanced a K(+) current which was linear and reversed close to 0 mV. Under physiological K(+) conditions, baclofen enhanced outward K(+) current and caused membrane hyperpolarisation, effects inhibited by 100 nM CGP 55845. Current enhancement was virtually abolished in the presence of 300 microM Zn(2+), a selective inhibitor of TASK-1. When recording membrane potential from MAH cells in clusters, hypoxic depolarisation was augmented by 100 nM CGP 55845. These data demonstrate that GABA(B) receptors mediate autoreceptor feedback in the adrenal medulla presumably via TASK-1, demonstrating a common autoregulatory feedback pathway in neurosecretory, chemosensitive cells.