Amy S. Miner

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 (IC50 = 0.5–5 μM) but is a poor inhibitor of purified turkey smooth muscle myosin II (IC50 ∼80 μM). We found that blebbistatin potently (IC50 ∼3 μM) 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. Blebbistatin also inhibited the KCl-induced tonic contractions produced by rabbit femoral and renal arteries that express primarily SMA and the weaker tonic contraction produced by the saphenous artery that expresses primarily SMB, with an equivalent potency comparable with that identified for nonmuscle myosin IIA (IC50 ∼5 μM). In femoral and saphenous arteries, blebbistatin had no effect on unloaded shortening velocity or the tonic increase in myosin light-chain phosphorylation produced by KCl but potently inhibited β-escin permeabilized artery contracted with calcium at pCa 5, suggesting that cell signaling events upstream from KCl-induced activation of cross-bridges were unaffected by blebbistatin. It is noteworthy that KCl-induced contractions of chicken gizzard were less potently inhibited (IC50 ∼20 μM). Adult femoral, renal, and saphenous arteries did not express significant levels of nonmuscle myosin. These data together indicate that blebbistatin is a potent inhibitor of smooth muscle myosin II, supporting the hypothesis that the force-bearing structure responsible for tonic force maintenance in adult mammalian vascular smooth muscle is the cross-bridge formed from the blebbistatin-dependent interaction between actin and smooth muscle myosin II.

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 μM indomethacin and ibuprofen (non‐selective COX inhibitors). SRC was concentration‐dependently inhibited by selective COX‐1 (SC‐560 and FR‐122047) and COX‐2 inhibitors (NS‐398 and LM‐1685), and by SC‐51089, a selective antagonist for the PGE‐2 receptor (EP) and ICI‐192,605 and SQ‐29,548, selective antagonists for thromboxane receptors (TP). The partial agonist/antagonist of the PGF‐2α receptor (FP), AL‐8810, inhibited SRC by ∼50%. Maximum inhibition was ∼90% by SC‐51089, ∼80–85% by the COX inhibitors and ∼70% by TP receptor antagonists. In the presence of ibuprofen to abolish SRC, PGE‐2, sulprostone, misoprostol, PGF‐2α and U‐46619 (thromboxane mimetic) caused rhythmic contractions that mimicked SRC. Fluorescence immunohistochemistry coupled with confocal laser scanning microscopy revealed that c‐Kit and vimentin co‐localized to interstitial cells surrounding detrusor smooth muscle bundles, indicating the presence of extensive ICCs in rabbit bladder. Co‐localization of COX‐1 and vimentin, and COX‐2 and vimentin by ICCs supports the hypothesis that ICCs were the predominant cell type in rabbit bladder expressing both COX isoforms. These data together suggest that ICCs appear to be an important source of prostaglandins that likely play a role in regulation of SRC. Additional studies on prostaglandin‐dependent SRC may generate opportunities for the application of novel treatments for disorders leading to overactive bladder.

Role of Protein Kinase Cζ 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 Ca2+ 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 PKCζ (PIPKCζ) inhibited KCl-induced tonic F. A myristoylated pseudosubstrate inhibitor of PKCα/β that inhibited phorbol dibutyrate-induced F slightly potentiated KCl-induced tonic F and attenuated 30 mM KCl-induced F. Although the ROCK inhibitor, H-1152 [(S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)-sulfonyl]-hexahydro-1H-1,4-diazepine dihydrochloride], reduced basal phosphorylation of myosin light-chain phosphatase-targeting subunit at Thr853 (MYPT1-pT853), 3 and 10 μM GF-109203X inhibited only KCl-stimulated phosphorylation, not basal MYPT1-pT853. In fura-2-loaded tissues, GF-109203X and PIPKCζ elevated basal [Ca2+]i (calcium) and potentiated KCl-induced tonic increases in calcium while reducing KCl-induced tonic increases in F. Blockade by nifedipine of Ca2+ entry through voltage-operated Ca2+ channels reduced KCl-induced Ca2+ sensitization and KCl-stimulated but not basal MYPT1-pT853. These data together support a model in which ROCK and PKCζ are constitutively active and function in “resting” muscle to regulate the basal levels of MYPT1-pT853 and calcium, respectively. In this model, KCl-induced increases in calcium activate PKCζ to feed forward and cause additional MYPT1-pT853 above that induced by constitutive ROCK, permitting Ca2+ sensitization and strong F maintenance. Active PKCζ also feeds back to attenuate the degree of KCl-induced increases in calcium.

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. Strips of rabbit DSM were quick-stretched (5 ms) and held isometric for 10 s to measure the resulting peak quick-stretch contractile response (PQSR). The ability of selective Ca(2+) channel blockers and kinase inhibitors to alter the PQSR was measured, and the phosphorylation levels of myosin light chain (MLC) and myosin phosphatase targeting regulatory subunit (MYPT1) were recorded. DSM responded to a quick-stretch with a biphasic response consisting of an initial contraction peaking at 0.24+/-0.02-fold the maximum KCl-induced contraction (F(o)) by 1.48+/-0.17 s (PQSR) before falling to a weaker tonic (10 s) level (0.12+/-0.03-fold F(o)). The PQSR was dependent on the rate and degree of muscle stretch, displayed a refractory period, and was converted to a sustained response in the presence of muscarinic receptor stimulation. The PQSR was inhibited by nifedipine, 2-aminoethoxydiphenyl borate (2-APB), 100 microM gadolinium and Y-27632, but not by atropine, 10 microM gadolinium, LOE-908, cyclopiazonic acid, or GF-109203X. Y-27632 and nifedipine abolished the increase in MLC phosphorylation induced by a quick-stretch. Y-27632, but not nifedipine, inhibited basal MYPT1 phosphorylation, and a quick-stretch failed to increase phosphorylation of this rhoA kinase (ROCK) substrate above the basal level. These data support the hypothesis that constitutive ROCK activity is required for a quick-stretch to activate Ca(2+) entry and cause a myogenic contraction of DSM.

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. The PKC inhibitor, GF-109203X (> or =3 microM) and the pseudo-substrate inhibitor of PKCzeta produced a response similar to BEL. BEL reduced basal and KCl-stimulated myosin phosphatase phosphorylation. Whereas BEL and H-1152 produced strong inhibition of KCl-induced tonic T (approximately 50%), H-1152 did not induce additional inhibition of tissues already inhibited by BEL, suggesting that iPLA(2) links KCl stimulation with ROCK activation. The cPLA(2) inhibitor, pyrrolidine-1, inhibited KCl-induced tonic increases in [Ca(2+)](i) but not T, whereas the inhibitor of 20-HETE production, HET0016, acted like the ROCK inhibitor H-1152 by causing Ca(2+) desensitization. These data support a model in which iPLA(2) activity regulates Ca(2+) sensitivity.

Failure of Bay K 8644 to induce RhoA kinase‐dependent calcium sensitization in rabbit blood vessels

Background and purpose:  RhoA kinase (ROCK) participates in K+ depolarization (KCl)‐induced Ca2+ sensitization of contraction. Whether constitutive, depolarization‐ or Ca2+‐activated ROCK plays the major role in this signalling system remains to be determined. Here, we determined whether Bay K 8644, a dihydropyridine that promotes Ca2+ channel clusters to operate in a persistent Ca2+ influx mode, could cause ROCK‐dependent Ca2+ sensitization.

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 sphingo‐lipid metabolite, sphingosine‐1‐phosphate (S1P), in regulating rabbit detrusor smooth muscle tone and con‐traction. S1P‐induced contraction of detrusor muscle was dependent on stretch and intracellular calcium. Although detrusor expresses the S1P receptors S1P1 and S1P2, only S1P2 appeared to be involved in S1P‐induced contraction, since SEW2871 (S1P1 agonist) and dihydro‐S1P (potent agonist for all S1P receptors except S1P2) were poor contractile agents. In agreement, the S1P2 antagonist JTE013 inhibited S1P‐induced contraction. The fast, transient muscle contraction (phasic) mediated by S1P was dependent on phospholipase C (PLC) whereas the slower, sustained contraction (tonic) was not. Surprisingly, the immunosuppressant FTY720‐phosphate, an agonist for all S1P receptors except S1P2, had distinct contractile properties and also induced slow, sustained contraction. Thus, FTY720‐phos‐phate and/or S1P may regulate calcium channels in an S1P receptor‐independent manner. Collectively, our results demonstrate that S1P may regulate detrusor smooth muscle tone and suggest that dysregulation of complex S1P signaling might contribute to OBS.—Watterson, K. R., Berg, K. M., Kapitonov, D., Payne, S. G., Miner, A. S., Bittman, R., Milstien, S., Ratz, P. H., Spiegel, S. Sphingosine‐1‐phosphate and the immuno‐suppressant, fty720‐phosphate, regulate detrusor muscle tone. FASEB J. 21, 2818–2828 (2007)

Potentiation of carbachol-induced detrusor smooth muscle contractions by β-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. The tyrosine kinase inhibitor, genistein, inhibited carbachol-induced contractions only in the presence of isoproterenol. Contraction produced by a single high carbachol concentration (1 microM) displayed 1 fast and 1 slow peak. In the presence of isoproterenol, the slow peak was not strengthened, but was delayed, and U-0126 (mitogen-activated protein kinase kinase inhibitor) selectively inhibited this delay concomitantly with inhibition of extracellular signal-regulated kinase (ERK) phosphorylation. Isoproterenol reduced ERK phosphorylation only in the absence of carbachol. These data support the concept that, by inhibiting weak contractions, potentiating strong contractions, and producing a more switch-like concentration-response curve, beta-adrenoceptor stimulation enhanced the effectiveness of muscarinic receptor-induced detrusor smooth muscle contraction. Moreover, beta-adrenoceptor stimulation changed the cellular mechanism by which carbachol produced contraction. The potential significance of multi-receptor and multi-cell crosstalk is discussed.

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 0.8-fold L(0). Typically, three repeated contractions were necessary for full length adaptation to occur. The tonic phase of a third KCl-induced contraction was increased by ∼50% after the release of tissues from 1.25-fold to 0.75-fold L(o). The mechanism for this phenomenon did not appear to lie in thick filament regulation because there was no increase in myosin light chain (MLC) phosphorylation to support the increase in tension nor was length adaptation abolished when Ca(2+) entry was limited by nifedipine and when Rho kinase (ROCK) was blocked by H-1152. However, length adaptation of both the phasic and tonic phases was abolished when actin polymerization was inhibited through blockade of the plus end of actin by cytochalasin-D. Interestingly, inhibition of actin polymerization when G-actin monomers were sequestered by latrunculin-B increased the phasic phase and had no effect on the tonic phase of contraction during length adaptation. These data suggest that for a given level of cytosolic free Ca(2+), active tension in the femoral artery can be sensitized not only by regulation of MLC phosphatase via ROCK and protein kinase C, as has been reported by others, but also by a nonmyosin regulatory mechanism involving actin polymerization. Dysregulation of this form of active tension modulation may provide insight into alterations of large artery stiffness in hypertension.

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]. Filling tone, however, increased in a linear fashion from 70% to 120% L(ref). In the presence of ibuprofen to abolish autonomous contraction and retain adjustable preload tension, tension was reduced in strength but remained linearly dependent on length from 70% to 120% L(ref). In the absence of autonomous contraction, stretching detrusor muscle from 80% to 120% L(ref) still caused an increase in tone during PGE(2)-induced rhythmic contraction, suggesting that muscle stretch caused increases in detrusor muscle contractile sensitivity rather than in prostaglandin release. In the absence of autonomous contraction, the degree of adjustable preload tension and myosin phosphorylation increased when detrusor was stretched from 80% to 120% L(ref), but also displayed length-hysteresis, indicating that detrusor muscle senses preload rather than muscle length. Together, these data support the hypothesis that detrusor muscle acts as a preload tension sensor. Because detrusor muscle is in-series with neuronal mechanosensors responsible for urinary urgency, a more thorough understanding of detrusor muscle filling tone may reveal unique targets for therapeutic intervention of contractile disorders such as overactive bladder.