The AMP-Dependent Protein Kinase (AMPK) Activator A-769662 Causes Arterial Relaxation by Reducing Cytosolic Free Calcium Independently of an Increase in AMPK Phosphorylation.

Although recent studies reveal that activation of the metabolic and Ca sensor AMPK strongly inhibits smooth muscle contraction, there is a paucity of information about the potential linkage between pharmacological AMPK activation and vascular smooth muscle (VSM) contraction regulation. Our aim was to test the general hypothesis that the allosteric AMPK activator A-769662 causes VSM relaxation via inhibition of contractile protein activation, and to specifically determine which activation mechanism(s) is(are) affected. The ability of A-769662 to cause endothelium-independent relaxation of contractions induced by several contractile stimuli was examined in large and small musculocutaneous and visceral rabbit arteries.

AICAR Administration Attenuates Hemorrhagic Hyperglycemia and Lowers Oxygen Debt in Anesthetized Male Rabbits.

Many strategies have been utilized to treat traumatic shock via improved oxygen delivery (DO), while fewer have been used to in an attempt to reduce oxygen demand (VO). The cellular energy sensor 5' adenosine monophosphate-activated protein kinase (AMPK) has the potential to modulate both whole-body DO and VO. Therefore, we determined the effect of the AMPK activator AICAR (5-aminoimidazole-4-carboxamide 1-β-D-ribonucleoside) given acutely or chronically on key metabolites, hemodynamics, and oxygen consumption/delivery before and during hemorrhage in anesthetized male rabbits.

Metabolic Stress-Induced Activation of AMPK and Inhibition of Constitutive Phosphoproteins Controlling Smooth Muscle Contraction: Evidence for Smooth Muscle Fatigue?

Metabolic stress diminishes smooth muscle contractile strength by a poorly defined mechanism. To test the hypothesis that metabolic stress activates a compensatory cell signaling program to reversibly downregulate contraction, arterial rings and bladder muscle strips were deprived of O and glucose for 30 and 60 min ("starvation") to induce metabolic stress, and the phosphorylation status of proteins involved in regulation of contraction and metabolic stress were assessed in tissues under basal and stimulated conditions. A 15-30 min recovery period (O and glucose repletion) tested whether changes induced by starvation were reversible.

Slowly cycling Rho kinase-dependent actomyosin cross-bridge "slippage" explains intrinsic high compliance of detrusor smooth muscle.

Biological soft tissues are viscoelastic because they display time-independent pseudoelasticity and time-dependent viscosity. However, there is evidence that the bladder may also display plasticity, defined as an increase in strain that is unrecoverable unless work is done by the muscle. In the present study, an electronic lever was used to induce controlled changes in stress and strain to determine whether rabbit detrusor smooth muscle (rDSM) is best described as viscoelastic or viscoelastic plastic.

Elevated steady-state bladder preload activates myosin phosphorylation: detrusor smooth muscle is a preload tension sensor.

In rabbit bladder wall (detrusor) muscle, the degree of tone induced during physiological filling (filling tone) is the sum of adjustable preload tension and autonomous contractile tension. The present study was designed to determine whether the level of filling tone is dependent on detrusor muscle length. Maximum active tension induced by KCl was parabolic in relation to length [tension increased from 70% to 100% of a reference length (L(ref)) and decreased at longer muscle lengths].

Rho-kinase inhibition attenuates calcium-induced contraction in β-escin but not Triton X-100 permeabilized rabbit femoral artery.

K+-depolarization (KCl) of smooth muscle has long been known to cause Ca2+-dependent contraction, but only recently has this G protein-coupled receptor (GPCR)-independent stimulus been associated with rhoA kinase (ROCK)-dependent myosin light chain (MLC) phosphatase inhibition and Ca2+ sensitization. This study examined effects of ROCK inhibition on the concentration-response curves (CRCs) generated in femoral artery by incrementally adding increasing concentrations of KCl to intact tissues, and Ca2+ to tissues permeabilized with Triton X-100, β-escin and α-toxin. For a comparison, tissue responses were assessed also in the presence of protein kinase C (PKC) and MLC kinase inhibition.

Prostaglandin E₂mediates spontaneous rhythmic contraction in rabbit detrusor muscle.

Introduction: The purpose of this investigation was to determine if prostaglandin E₂(PGE₂) is produced by rabbit detrusor free of urothelium and demonstrate that PGE₂ is responsible for the generation of spontaneous rhythmic contraction (SRC).

Methods: A bioassay was performed in which contraction frequency in strips of rabbit detrusor was compared before and after addition of superfusate from incubating sections of rabbit detrusor. Specificity was determined by testing the effects of SC-51089, a PGE₂(EP1) antagonist.

Active tension adaptation at a shortened arterial muscle length: inhibition by cytochalasin-D.

Unlike the static length-tension curve of striated muscle, airway and urinary bladder smooth muscles display a dynamic length-tension curve. Much less is known about the plasticity of the length-tension curve of vascular smooth muscle. The present study demonstrates that there were significant increases of ∼15% in the phasic phase and ∼10% in the tonic phase of a third KCl-induced contraction of a rabbit femoral artery ring relative to the first contraction after a 20% decrease in length from an optimal muscle length (L(0)) to 0.

Calcium-independent phospholipase A2 participates in KCl-induced calcium sensitization of vascular smooth muscle.

In vascular smooth muscle, KCl not only elevates intracellular free Ca(2+) ([Ca(2+)](i)), myosin light chain kinase activity and tension (T), but also can inhibit myosin light chain phosphatase activity by activation of rhoA kinase (ROCK), resulting in Ca(2+) sensitization (increased T/[Ca(2+)](i) ratio). Precisely how KCl causes ROCK-dependent Ca(2+) sensitization remains to be determined. Using Fura-2-loaded isometric rings of rabbit artery, we found that the Ca(2+)-independent phospholipase A(2) (iPLA(2)) inhibitor, bromoenol lactone (BEL), reduced the KCl-induced tonic but not early phasic phase of T and potentiated [Ca(2+)](i), reducing Ca(2+) sensitization.

Potentiation of carbachol-induced detrusor smooth muscle contractions by beta-adrenoceptor activation.

In strips of rabbit bladder free of urothelium, the beta-adrenoceptor agonist, isoproterenol, significantly reduced basal detrusor smooth muscle tone and inhibited contractions produced by low concentrations of the muscarinic receptor agonist, carbachol. During a carbachol concentration-response curve, instead of inhibiting, isoproterenol strengthened contractions produced by high carbachol concentrations. Thus, the carbachol concentration-response curve was shifted by isoproterenol from a shallow, graded relationship, to a steep, switch-like relationship.

Potential for control of detrusor smooth muscle spontaneous rhythmic contraction by cyclooxygenase products released by interstitial cells of Cajal.

Interstitial cells of Cajal (ICCs) have been identified as pacemaker cells in the upper urinary tract and urethra, but the role of ICCs in the bladder remains to be determined. We tested the hypotheses that ICCs express cyclooxygenase (COX), and that COX products (prostaglandins), are the cause of spontaneous rhythmic contraction (SRC) of isolated strips of rabbit bladder free of urothelium. SRC was abolished by 10 microM indomethacin and ibuprofen (non-selective COX inhibitors).

Role of protein kinase Czeta and calcium entry in KCl-induced vascular smooth muscle calcium sensitization and feedback control of cellular calcium levels.

The degree of tonic force (F) maintenance induced in vascular smooth muscle upon K(+) depolarization with 110 mM KCl can be greatly reduced by inhibition of rhoA kinase (ROCK). We explored the possibility that a protein kinase C (PKC) isotype may also play a role in causing KCl-induced Ca(2+) sensitization. In isometric rings of rabbit artery, the PKC inhibitors, Go-6983 (3-[1-[3-(dimethylamino)propyl]-5-methoxy-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione), GF-109203X (2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl) maleimide), and a cell-permeable (myristoylated) pseudosubstrate inhibitor of PKCzeta (PI(PKCzeta)) inhibited KCl-induced tonic F.

Stimulated calcium entry and constitutive RhoA kinase activity cause stretch-induced detrusor contraction.

Urinary bladder wall muscle (i.e., detrusor smooth muscle; DSM) contracts in response to a quick-stretch, but this response is neither fully characterized, nor completely understood at the subcellular level.

Sphingosine-1-phosphate and the immunosuppressant, FTY720-phosphate, regulate detrusor muscle tone.

Overactive bladder syndrome (OBS) results from disturbances of bladder function. Bladder smooth muscle (detrusor) exhibits spontaneous rhythmic activity (tone) independent of neurogenic control, which is enhanced in patients with OBS. We have now uncovered a prominent role for the bioactive sphingolipid metabolite, sphingosine-1-phosphate (S1P), in regulating rabbit detrusor smooth muscle tone and contraction.

Potent inhibition of arterial smooth muscle tonic contractions by the selective myosin II inhibitor, blebbistatin.

Blebbistatin is reported to be a selective and specific small molecule inhibitor of the myosin II isoforms expressed by striated muscles and nonmuscle (IC(50) = 0.5-5 microM) but is a poor inhibitor of purified turkey smooth muscle myosin II (IC(50) approximately 80 microM). We found that blebbistatin potently (IC(50) approximately 3 microM) inhibited the actomyosin ATPase activities of expressed "slow" [smooth muscle myosin IIA (SMA)] and "fast" [smooth muscle myosin IIB (SMB)] smooth muscle myosin II heavy-chain isoforms.

Evidence for absence of latch-bridge formation in muscular saphenous arteries.

Large-diameter elastic arteries can produce strong contractions indefinitely at a high-energy economy by the formation of latch bridges. Whether downstream blood vessels also use latch bridges remains unknown. The zero-pressure medial thickness and lumen diameter of rabbit saphenous artery (SA), a muscular branch of the elastic femoral artery (FA), were, respectively, approximately twofold and half-fold that of the FA.

Convergence of Ca2+-desensitizing mechanisms activated by forskolin and phenylephrine pretreatment, but not 8-bromo-cGMP.

Contractile stimuli can sensitize myosin to Ca2+ by activating RhoA kinase (ROK) and PKC that inhibit myosin light chain phosphatase (MLCP) activity. Relaxant stimuli, acting through PKA and PKG (cyclic nucleotide-dependent protein kinases), and pretreatment with contractile agents such as phenylephrine (PE), can desensitize myosin to Ca2+. It is unknown precisely how these stimuli cause Ca2+ desensitization.

Regulation of smooth muscle calcium sensitivity: KCl as a calcium-sensitizing stimulus.

KCl has long been used as a convenient stimulus to bypass G protein-coupled receptors (GPCR) and activate smooth muscle by a highly reproducible and relatively "simple" mechanism involving activation of voltage-operated Ca2+ channels that leads to increases in cytosolic free Ca2+ ([Ca2+]i), Ca2+-calmodulin-dependent myosin light chain (MLC) kinase activation, MLC phosphorylation and contraction. This KCl-induced stimulus-response coupling mechanism is a standard tool-set used in comparative studies to explore more complex mechanisms generated by activation of GPCRs. One area where this approach has been especially productive is in studies designed to understand Ca2+ sensitization, the relationship between [Ca2+]i and force produced by GPCR agonists.

Length-dependent regulation of basal myosin phosphorylation and force in detrusor smooth muscle.

Urinary bladder (detrusor) smooth muscle is active in the absence of an external stimulus. Tone occurs even "at rest" during the filling phase, and it is elevated in patients with overactive bladder. This study examined the role of muscle length on tone and the level of basal myosin light chain phosphorylation (MLC(20P)).