PKC activates BKCa channels in rat pulmonary arterial smooth muscle via cGMP-dependent protein kinase

Normally, signaling mechanisms that activate large-conductance, calcium- and voltage-activated potassium (BK(Ca)) channels in pulmonary vascular smooth muscle cause pulmonary vasodilatation. BK(Ca)-channel modulation is important in the regulation of pulmonary arterial pressure, and inhibition (decrease in the opening probability) of the BK(Ca) channel has been implicated in the development of pulmonary vasoconstriction. Protein kinase C (PKC) causes pulmonary vasoconstriction, but little is known about the effect of PKC on BK(Ca)-channel activity in pulmonary vascular smooth muscle. Accordingly, studies were done to determine the effect of PKC on BK(Ca)-channel activity using patch-clamp studies in pulmonary arterial smooth muscle cells (PASMCs) of the Sprague-Dawley rat. The PKC activators phorbol myristate acetate (PMA) and thymeleatoxin opened BK(Ca) channels in single Sprague-Dawley rat PASMC. The activator response to both PMA and thymeleatoxin on BK(Ca)-channel activity was blocked by G?-6983, which selectively blocks PKC-alpha, -delta, -gamma, and -zeta, and by rottlerin, which selectively inhibits PKC-delta. In addition, the specific cyclic GMP-dependent protein kinase antagonist KT-5823 blocked the responses to PMA and thymelatoxin, whereas the specific cyclic AMP-dependent protein kinase blocker KT-5720 had no effect. In isolated pulmonary arterial vessels, both PMA and forskolin caused vasodilatation, which was inhibited by KT-5823, G?-6983, or the BK(Ca)-channel blocker tetraethylammonium. The results of this study indicate that activation of specific PKC isozymes increases BK(Ca)-channel activity in Sprague-Dawley rat PASMC via cyclic GMP-dependent protein kinase, which suggests a unique signaling mechanism for vasodilatation.     

Basic residues of the helix six domain of influenza virus M1 involved in nuclear translocation of M1 can be replaced by PTAP and YPDL late assembly domain motifs

Influenza type A virus matrix (M1) protein possesses multiple functional motifs in the helix 6 (H6) domain (amino acids 91 to 105), including nuclear localization signal (NLS) (101-RKLKR-105) involved in translocating M1 from the cytoplasm into the nucleus. To determine the role of the NLS motif in the influenza virus life cycle, we mutated these and the neighboring sequences by site-directed mutagenesis, and influenza virus mutants were generated by reverse genetics. Our results show that infectious viruses were rescued by reverse genetics from all single alanine mutations of amino acids in the H6 domain and the neighboring region except in three positions (K104A and R105A within the NLS motif and E106A in loop 6 outside the NLS motif). Among the rescued mutant viruses, R101A and R105K exhibited reduced growth and small-plaque morphology, and all other mutant viruses showed the wild-type phenotype. On the other hand, three single mutations (K104A, K105A, and E106A) and three double mutations (R101A/K102A, K104A/K105A, and K102A/R105A) failed to generate infectious virus. Deletion (Delta YRKL) or mutation (4A) of YRKL also abolished generation of infectious virus. However, replacement of the YRKL motif with PTAP or YPDL as well as insertion of PTAP after 4A mutation yielded infectious viruses with the wild-type phenotype. Furthermore, mutant M1 proteins (R101A/K102A, Delta YRKL, 4A, PTAP, 4A+PTAP, and YPDL) when expressed alone from cloned cDNAs were only cytoplasmic, whereas the wild-type M1 expressed alone was both nuclear and cytoplasmic as expected. These results show that the nuclear translocation function provided by the positively charged residues within the NLS motif does not play a critical role in influenza virus replication. Furthermore, these sequences of H6 domain can be replaced by late (L) domain motifs and therefore may provide a function similar to that of the L domains of other negative-strand RNA and retroviruses.     

At physiological temperatures the ATPase rates of shortening soleus and psoas myofibrils are similar

We obtained the temperature dependences of the adenosine triphosphatase (ATPase) activities (calcium-activated and relaxed) of myofibrils from a slow muscle, which we compared with those from a fast muscle. We chose rabbit soleus and psoas because their myosin heavy chains are almost pure: isoforms I and IIX, respectively. The Arrhenius plots of the ATPases are linear (4-35 degrees C) with energies of activation for soleus myofibrils 155 kJ mol(-1) (activated) and 78 kJ mol(-1) (relaxed). With psoas myofibrils, the energies of activation were 71 kJ mol(-1) (activated) and 60 kJ mol(-1) (relaxed). When extrapolated to 42 degrees C the ATPase rates of the two types of myofibril were identical: 50 s(-1) (activated) and 0.23 s(-1) (relaxed). Whereas with psoas myofibrils the K(m) for adenosine triphosphate (activated ATPase) is relatively insensitive to temperature, that for soleus myofibrils increased from 0.3 microM at 4 degrees C to 66.5 microM at 35 degrees C. Our results illustrate the importance of temperature when comparing the mechanochemical coupling in different types of muscle. We discuss the problem of how to reconcile the similarity of the myofibrillar ATPase rates at physiological temperatures with their different mechanical properties.     

Evidence that phosphate release is the rate-limiting step on the overall ATPase of psoas myofibrils prevented from shortening by chemical cross-linking

It has been suggested that the mechanical condition determines the rate-limiting step of the ATPase of the myosin heads in fibers: when fibers are isometrically contracting, the ADP release kinetics are rate-limiting, but as the strain is reduced and the fibers are allowed to shorten, the ADP release kinetics accelerate and P(i) release becomes rate-limiting. We have put this idea to the test with myofibrils as a model because with these both mechanical and chemical kinetic measurements are possible. With relaxed or rapidly shortening myofibrils, P(i) release is rate-limiting and (A)M.ADP.P(i) states accumulate in the steady state [Lionne, C., et al. (1995) FEBS Lett. 364, 59]. We have now studied the kinetics of P(i) release with chemically cross-linked myofibrils that, when adequately cross-linked, appear to be a good model for isometric contraction. By using a method that is specific for free P(i) and rapid quench flow that measures the amount of (A)M.ADP.P(i) states and free P(i), we show that (A)M.ADP.P(i) states predominate which suggests that the overall ATPase is limited by P(i) release kinetics. Therefore, under our experimental conditions with myofibrils prevented from shortening, the concentration of (A)M.ADP states is low, as with rapidly shortening and relaxed myofibrils. This result is difficult to reconcile with the sensitivity of force development in fibers and myofibrils to P(i) which implies interaction of P(i) with an (A)M.ADP state. We discuss two models for accommodating the mechanical and chemical kinetics with reference to the duty cycle in skeletal muscle.     

Defenselike patterns of spinal sympathetic outflow involving the 10-Hz and cardiac-related rhythms

Frequency- and time-domain analyses were used to compare the effects of stimulation of the defense region of the midbrain periaqueductal gray (PAG) on the 10-Hz and cardiac-related discharges of sympathetic nerves with different cardiovascular targets. In baroreceptor-denervated cats anesthetized with urethan, PAG stimulation at frequencies equal to or higher (up to 25 Hz) than that of the free-running 10-Hz rhythm produced an immediate and sustained decrease in vertebral sympathetic nerve (VN) 10-Hz activity but increased the 10-Hz discharges of the inferior cardiac (CN) and renal (RN) nerves. In baroreceptor-innervated cats, VN cardiac-related activity was initially unchanged by high-frequency (25-Hz) PAG stimulation, or it increased along with that in the CN and RN. Later, during high-frequency PAG stimulation, when the rise in blood pressure approached its peak, VN cardiac-related activity usually was reduced below control level. At this time, the increases in CN and RN cardiac-related discharges were largely sustained. The cardiac-related discharges of the three nerves were unaffected by PAG stimulation at frequencies just below or just above that of the heartbeat. We conclude that the defenselike pattern of spinal sympathetic outflow involving the 10-Hz rhythm is different in mechanism and character from that involving the cardiac-related rhythm.     

Differential pattern of spinal sympathetic outflow in response to stimulation of the caudal medullary raphe

In urethan-anesthetized cats, frequency domain analysis was used to explore the mechanisms of differential responses of inferior cardiac (CN), vertebral (VN), and renal (RN) sympathetic nerves to electrical stimulation of a discrete region of the medullary raphe (0-2 mm caudal to the obex). Raphe stimulation in baroreceptor-denervated cats at frequencies (7-12 Hz) that entrained the 10-Hz rhythm in nerve activity decreased CN and RN activities but increased VN activity. The reductions in CN and RN discharges were associated with decreased low-frequency (相似文献   

Vasoconstrictor effect of endothelin-1 on hypertensive pulmonary arterial smooth muscle involves Rho-kinase and protein kinase C

Although one of the common characteristics of pulmonary hypertension is abnormal sustained vasoconstriction, the signaling pathways that mediate this heightened pulmonary vascular response are still not well defined. Protein kinase C (PKC) and Rho-kinase are regulators of smooth muscle contraction induced by G protein-coupled receptor agonists including endothelin-1 (ET-1), which has been implicated as a signaling pathway in pulmonary hypertension. Toward this end, it was hypothesized that both Rho-kinase and PKC mediate the pulmonary vascular response to ET-1 in hypertensive pulmonary arterial smooth muscle, and therefore, the purpose of this study was to determine the role of PKC and Rho-kinase signaling in ET-1-induced vasoconstriction in both normotensive (Sprague-Dawley) and hypertensive (Fawn-Hooded) rat pulmonary arterial smooth muscle. Results indicate that ET-1 caused greater vasoconstriction in hypertensive pulmonary arteries compared with the normal vessels, and treatment with the PKC antagonists chelerythrine, rottlerin, and G? 6983 inhibited the vasoconstrictor response to ET-1 in the hypertensive vessels. In addition, the specific Rho-kinase inhibitor Y-27632 significantly attenuated the effect of ET-1 in both normotensive and hypertensive phenotypes, with greater inhibition occurring in the hypertensive arteries. Furthermore, Western blot analysis revealed that ET-1 increased RhoA expression in both normotensive and hypertensive pulmonary arteries, with expression being greater in the hypertensive state. These results suggest that both PKC and Rho/Rho-kinase mediate the heightened pulmonary vascular response to ET-1 in hypertensive pulmonary arterial smooth muscle.     

Loss of intramolecular electrostatic interactions and limited conformational ensemble may promote self‐association of cis–tau peptide

Self‐association of proteins can be triggered by a change in the distribution of the conformational ensemble. Posttranslational modification, such as phosphorylation, can induce a shift in the ensemble of conformations. In the brain of Alzheimer's disease patients, the formation of intra‐cellular neurofibrillary tangles deposition is a result of self‐aggregation of hyper‐phosphorylated tau protein. Biochemical and NMR studies suggest that the cis peptidyl prolyl conformation of a phosphorylated threonine‐proline motif in the tau protein renders tau more prone to aggregation than the trans isomer. However, little is known about the role of peptidyl prolyl cis/trans isomerization in tau aggregation. Here, we show that intra‐molecular electrostatic interactions are better formed in the trans isomer. We explore the conformational landscape of the tau segment containing the phosphorylated‐Thr²³¹‐Pro²³² motif using accelerated molecular dynamics and show that intra‐molecular electrostatic interactions are coupled to the isomeric state of the peptidyl prolyl bond. Our results suggest that the loss of intra‐molecular interactions and the more restricted conformational ensemble of the cis isomer could favor self‐aggregation. The results are consistent with experiments, providing valuable complementary atomistic insights and a hypothetical model for isomer specific aggregation of the tau protein. Proteins 2015; 83:436–444. © 2014 Wiley Periodicals, Inc.     

Perturbation of yeast 3-phosphoglycerate kinase reaction mixtures with ADP: transient kinetics of formation of ATP from bound 1,3-bisphosphoglycerate

3-Phosphoglycerate kinase (PGK) is the first ATP-producing enzyme in glycolysis: ADP + 1,3-bisphosphoglycerate (bPG) <--> ATP + 3-phosphoglycerate (PG). Whereas extensive studies have been carried out on its structure, there is less information about its reaction pathway, which is usually studied in the reverse direction because of the instability of bPG. We studied the transients of the PGK reaction by chemical sampling in a rapid quench flow apparatus, using [gamma-(32)P]ATP, in 30% methanol at 4 degrees C to decrease k(cat). There were two types of experiment, both at low PG concentrations to prevent bPG release. In the first, reaction mixtures were quenched in acid at different times (from 4 ms) and the bPG concentrations were determined. This type gave information about the ATP binding and phospho-transfer steps. In the second, PGK reaction mixtures at equilibrium were perturbed by the injection of ADP, the new mixtures aged for different times and quenched in acid, and the bPG concentrations were determined. This gave information about the kinetics of the binding of ADP to a PGK intermediate. The data from the two types of experiments were fitted to simple schemes and then treated together by a global fitting procedure using a five-step pathway, deduced from previous structural studies. Under our conditions, it appears that (1) a binary PGK.bPG complex is an important intermediate on the reaction pathway, i.e., that ADP is released before bPG, (2) ADP binds to a "closed" conformation in the PGK.bPG complex, and (3) the PGK reaction can be studied in the physiologically important direction without having to handle bPG.     

Histone acetyltransferase 1 is dispensable for replication-coupled chromatin assembly but contributes to recover DNA damages created following replication blockage in vertebrate cells

Histone acetyltransferase 1 (HAT1) is implicated for diacetylation of Lys-5 and Lys-12 of newly synthesized histone H4, the biological significance of which remains unclear. To investigate the in vivo role of HAT1, we generated HAT1-deficient DT40 clone (HAT1(-/-)). HAT1(-/-) cells exhibited greatly reduced diacetylation levels of Lys-5 and Lys-12, and acetylation level of Lys-5 of cytosolic and chromatin histones H4, respectively. The in vitro nucleosome assembly assay and in vivo MNase digestion assay revealed that HAT1 and diacetylation of Lys-5 and Lys-12 of histone H4 are dispensable for replication-coupled chromatin assembly. HAT1(-/-) cells had mild growth defect, conferring sensitivities to methyl methanesulfonate and camptothecin that enforce replication blocks creating DNA double strand breaks. Such heightened sensitivities were associated with prolonged late-S/G2 phase. These results indicate that HAT1 participates in recovering replication block-mediated DNA damages, probably through chromatin modulation based on acetylation of Lys-5 and Lys-12 of histone H4.