Avril V. Somlyo

Signal transduction by G-proteins, rho-kinase and protein phosphatase to smooth muscle and non-muscle myosin II

We here review mechanisms that can regulate the activity of myosin II, in smooth muscle and non‐muscle cells, by modulating the Ca2+ sensitivity of myosin regulatory light chain (RLC) phosphorylation. The major mechanism of Ca2+ sensitization of smooth muscle contraction and non‐muscle cell motility is through inhibition of the smooth muscle myosin phosphatase (MLCP) that dephosphorylates the RLC in smooth muscle and non‐muscle. The active, GTP‐bound form of the small GTPase RhoA activates a serine/threonine kinase, Rho‐kinase, that phosphorylates the regulatory subunit of MLCP and inhibits phosphatase activity. G‐protein‐coupled release of arachidonic acid may also contribute to inhibition of MLCP acting, at least in part, through the Rho/Rho‐kinase pathway. Protein kinase C(s) activated by phorbol esters and diacylglycerol can also inhibit MLCP by phosphorylating and thereby activating CPI‐17, an inhibitor of its catalytic subunit; this mechanism is independent of the Rho/Rho‐kinase pathway and plays only a minor, transient role in the G‐protein‐coupled mechanism of Ca2+ sensitization. Ca2+ sensitization by the Rho/Rho‐kinase pathway contributes to the tonic phase of agonist‐induced contraction in smooth muscle, and abnormally increased activation of myosin II by this mechanism is thought to play a role in diseases such as high blood pressure and cancer cell metastasis.

Smooth muscle differentiation marker gene expression is regulated by Rhoa-mediated actin polymerization

Smooth muscle cell (SMC) differentiation is regulated by a complex array of local environmental cues, but the intracellular signaling pathways and the transcription mechanisms that regulate this process are largely unknown. We and others have shown that serum response factor (SRF) contributes to SMC-specific gene transcription, and because the small GTPase RhoA has been shown to regulate SRF, the goal of the present study was to test the hypothesis that RhoA signaling is a critical mechanism for regulating SMC differentiation. Coexpression of constitutively active RhoA in rat aortic SMC cultures significantly increased the activity of the SMC-specific promoters, SM22 and SM α-actin, whereas coexpression of C3 transferase abolished the activity of these promoters. Inhibition of either stress fiber formation with the Rho kinase inhibitor Y-27632 (10 μm) or actin polymerization with latrunculin B (0.5 μm) significantly decreased the activity of SM22 and SM α-actin promoters. In contrast, increasing actin polymerization with jasplakinolide (0.5 μm) increased SM22 and SM α-actin promoter activity by 22-fold and 13-fold, respectively. The above interventions had little or no effect on the transcription of an SRF-dependent c-fos promoter or on a minimal thymidine kinase promoter that is not SRF-dependent. Taken together, the results of these studies indicate that in SMC, RhoA-dependent regulation of the actin cytoskeleton selectively regulates SMC differentiation marker gene expression by modulating SRF-dependent transcription. The results also suggest that RhoA signaling may serve as a convergence point for the multiple signaling pathways that regulate SMC differentiation.

Quantitative electron probe microanalysis of biological thin sections: Methods and validity

Abstract A quantitative method of electron probe X-ray analysis of thin sections proposed by Hall [1] and based on the relationship between characteristic peak and X-ray continuum was evaluated in conjunction with a multiple least squares computer fitting program designed for the subtraction of peak from background counts and for the deconvolution of overlapping peaks. The theory of this quantitation is discussed including the effects of higher Z elements in normally low Z biological matrices. Estimates of the inherent statistical errors of the deconvolution program and the extent of systematic errors introduced by changes in detector resolution and energy calibration are given. The minimal detectable concentration of K in a typical biological thin section and using probe currents readily tolerated by the specimen over a 100 sec counting time were found to be approximately 10 mmol/kg. The minimal detectable mass in a 100 sec collection time using a thermionic gun was calculated to be approximately 10 −19 g in agreement with previous analysis of the iron core of single ferritin molecules [7]. The quantitation of Na and K in thin polymer sections is illustrated and the use of biological materials containing covalently bound elements as standards is described. Quantitation of P and Mg of such specimens gave results within 10% of chemical analysis. The effects of the transmission function of the detection system, ionization cross section and fluorescence yield on the X-ray counts generated by different elements are evaluated by analyzing binary standards and fitting the calculated transmission function to the X-ray continuum. There was excellent agreement between the experimentally determined continuum shape, the calculated transmission function and the results obtained with binary standards. The continuum followed the behavior expected of thin organic sections up to 1 ∥micron. Above this thickness the absorption of the low energy continuum within the specimen was observed. The effects of contamination and mass loss due to radiation damage were evaluated by measuring X-ray continuum and characteristic peaks as a function of electron dose. Contamination was proportional to probe current and inversely proportional to probe diameter. The mass loss due to radiation damage, as measured by X-ray continuum counts, was significantly different in serum albumin and in sucrose specimens. There was a 50% loss of sucrose mass at a dose of 0.03 C/cm 2 at room temperature. An Arrhenius plot of the process gave a thermal activation energy of 900 K. We interpret this finding to indicate that the mass loss is primarily due to vaporization or diffusion of small organic fragments. Serum albumin films showed 13% mass loss at doses up to 760 C/cm 2 . The sources of extraneous, other than the specimen, continuum were evaluated and characterized. It is shown that such contributions to the continuum can be recognized as originating from a thick target X-ray spectrum (specimen grid and holder, electron optical column) or by a low energy profile that is inconsistent with the X-ray transmission function of the beryllium window of the detector (scattered electron spectrum). Methods for correcting for such extraneous contributions to the continuum are given, and the optimum region (lowest error specimen continuum) for quantitation is evaluated. Examples of quantitative analysis of cryosections of human red blood cells, frog striated and rabbit smooth muscle are given and shown to be, within the limits of statistical error, in agreement with the results of chemical analyses. The total theoretically attainable improvement in minimal mass detection is estimated to be a factor of approximately 300, suggesting a detectable minimal mass of 10 −22 g. We conclude that quantitative analysis of ultrathin biological sections with a spatial resolution of at least 2000 A and an absolute accuracy of approximately 10% is feasible with the method described.

The effects of the Rho‐kinase inhibitor Y‐27632 on arachidonic acid‐, GTPγS‐, and phorbol ester‐induced Ca2+‐sensitization of smooth muscle

The effects of the Rho‐kinase inhibitor, Y‐27632 [1] on Ca2+‐sensitization of force induced by arachidonic acid (AA), phorbol 12,13‐dibutyrate (PDBu), GTPγS, and by the stable thromboxane analog, 9,11‐dideoxy‐9α,11α‐methanoepoxy‐PGF2α (U‐46619), were determined in α‐toxin‐permeabilized smooth muscles. Y‐27632 relaxed (up to 99%) Ca2+‐sensitization by GTPγS (10 μM) and U‐46619 (1 μM), but not by PDBu (20 μM), and reduced GTPγS‐induced myosin light chain (MLC20) phosphorylation from 28% to 17% (P=0.002). GTPγS‐induced force sensitization was inhibited by Y‐27632 more potently when the inhibitor was added during the plateau of force than prior to stimulation. In α‐toxin‐permeabilized smooth muscle, Y‐27632 inhibited AA (50 μM)‐induced Ca2+‐sensitization of force (by 66±1.3%) and reduced MLC20 phosphorylation. In contrast, Y‐27632 did not relax force Ca2+‐sensitized by AA in smooth muscle permeabilized with Triton X‐100. We conclude that (i) AA induces Ca2+‐sensitization through dual mechanisms, one mediated by Rho‐kinase (or a related kinase), and (ii) Rho‐kinase is not required for phorbol ester‐induced Ca2+‐sensitization.

Identification of the endogenous smooth muscle myosin phosphatase-associated kinase

Ca2+ sensitization of smooth muscle contraction involves inhibition of myosin light chain phosphatase (SMPP-1M) and enhanced myosin light chain phosphorylation. Inhibition of SMPP-1M is modulated through phosphorylation of the myosin targeting subunit (MYPT1) by either Rho-associated kinase (ROK) or an unknown SMPP-1M-associated kinase. Activated ROK is predominantly membrane-associated and its putative substrate, SMPP-1M, is mainly myofibrillar-associated. This raises a conundrum about the mechanism of interaction between these enzymes. We present ZIP-like kinase, identified by “mixed-peptide” Edman sequencing after affinity purification, as the previously unidentified SMPP-1M-associated kinase. ZIP-like kinase was shown to associate with MYPT1 and phosphorylate the inhibitory site in intact smooth muscle. Phosphorylation of ZIP-like kinase was associated with an increase in kinase activity during carbachol stimulation, suggesting that the enzyme may be a terminal member of a Ca2+ sensitizing kinase cascade.

Pharmacomechanical coupling: the role of calcium, G-proteins, kinases and phosphatases.

The concept of pharmacomechanical coupling, introduced 30 years ago to account for physiological mechanisms that can regulate contraction of smooth muscle independently of the membrane potential, has since been transformed from a definition into what we now recognize as a complex of well-defined, molecular mechanisms. The release of Ca2+ from the SR by a chemical messenger, InsP3, is well known to be initiated not by depolarization, but by agonist-receptor interaction. Furthermore, this G-protein-coupled phosphatidylinositol cascade, one of many processes covered by the umbrella of pharmacomechanical coupling, is part of complex and general signal transduction mechanisms also operating in many non-muscle cells of diverse organisms. It is also clear that, although the major contractile regulatory mechanism of smooth muscle, phosphorylation/dephosphorylation of MLC20, is [Ca2+]-dependent, the activity of both the kinase and the phosphatase can also be modulated independently of [Ca2+]i. Sensitization to Ca2+ is attributed to inhibition of SMPP-1M, a process most likely dominated by activation of the monomeric GTP-binding protein RhoA that, in turn, activates Rho-kinase that phosphorylates the regulatory subunit of SMPP-1M and inhibits its myosin phosphatase activity. It is likely that the tonic phase of contraction activated by a variety of excitatory agonists is, at least in part, mediated by this Ca(2+)-sensitizing mechanism. Desensitization to Ca2+ can occur either through inhibitory phosphorylation of MLCK by other kinases or autophosphorylation and by activation of SMPP-1M by cyclic nucleotide-activated kinases, probably involving phosphorylation of a phosphatase activator. Based on our current understanding of the complexity of the many cross-talking signal transduction mechanisms that operate in cells, it is likely that, in the future, our current concepts will be refined, additional mechanisms of pharmacomechanical coupling will be recognized, and those contributing to the pathologenesis diseases, such as hypertension and asthma, will be identified.

The contractile apparatus of vascular smooth muscle: Intermediate high voltage stereo electron microscopy

Intermediate high voltage stereo electron microscopy of rabbit portal-anterior mesenteric vein smooth muscle showed that thick filaments are 2·2 μm long and have tapered ends. In serial transverse sections, groups of three to five thick filaments end or begin within the same section. The intermediate (10 nm diameter) filaments were associated with dense bodies, as noted in previous studies, and were never seen within the substructure of the thick filaments. Actin filaments were found inserting on both cytoplasmic and plasma membrane-bound dense bodies. The greater length of the thick filaments in smooth than in striated muscles and their “parallel arrangement” within the fiber could contribute to the ability of smooth muscle to develop equal or greater tension than striated muscle, in spite of the much lower concentration of myosin in smooth muscle.

Release and recycling of calcium by the sarcoplasmic reticulum in guinea-pig portal vein smooth muscle.

The amplitude of interrupted contractions evoked by noradrenaline or caffeine in Ca2+‐free, high‐K+ solutions containing EGTA or La3+ was determined in small (40‐60 micron thick) bundles of guinea‐pig portal anterior mesenteric vein. Interrupted contractions were produced by removing the stimulating agent as soon as the amplitude of the tension record reached its peak. The distribution of intracellular Ca2+ was determined, with electron probe X‐ray microanalysis, in cryosections of preparations frozen in the relaxed state and at the peak of noradrenaline‐induced contractions. Interrupted contractions of maximal or near‐maximal amplitudes could be evoked every 2 min for up to 15 min in the virtual absence of extracellular Ca2+. If noradrenaline was allowed to remain in the solution throughout the period of spontaneous relaxation, a subsequent contraction could no longer be evoked in the absence of extracellular Ca2+. Interrupted contractions, similar to those evoked by noradrenaline, could also be stimulated by caffeine. The amplitude of reproducible interrupted contractions in Ca2+‐free, high‐K+ solution was graded with noradrenaline concentration. The ability of these smooth muscles to contract repeatedly and maximally in Ca2+‐free solutions indicates the recycling of Ca2+ released from an intracellular store. The occurrence of these contractions in high‐K+ (depolarizing) solutions supports the conclusion (Devine, Somlyo & Somlyo, 1972) that the release of intracellular Ca2+ is one of the mechanisms of pharmacomechanical coupling. The number of subplasmalemmal regions in which high Ca concentrations (greater than 10 mmol/kg dry wt.) were detected, with approximately 75 nm diameter electron probes, was reduced in muscles frozen at the peak of contraction, from 4.7/cell periphery in the relaxed to 1.4/cell periphery in the contracted preparations. In freeze‐substituted smooth muscles, in which the membranes of the junctional sarcoplasmic reticulum could be visualized, the regions containing high Ca were identified as part of the sarcoplasmic reticulum (s.r.), indicating that the s.r. is the store from which noradrenaline and caffeine release Ca2+.

L-type Voltage-Gated Ca2+ Channels Modulate Expression of Smooth Muscle Differentiation Marker Genes via a Rho Kinase/Myocardin/SRF–Dependent Mechanism

Vascular smooth muscle cell (SMC) contraction is mediated in part by calcium influx through L-type voltage-gated Ca2+ channels (VGCC) and activation of the RhoA/Rho kinase (ROK) signaling cascade. We tested the hypothesis that Ca2+ influx through VGCCs regulates SMC differentiation marker expression and that these effects are dependent on RhoA/ROK signaling. Depolarization-induced activation of VGCCs resulted in a nifedipine-sensitive increase in endogenous smooth muscle myosin heavy chain (SMMHC) and SM &agr;-actin expression and CArG-dependent promoter activity, as well as c-fos promoter activity. The ROK inhibitor, Y-27632, prevented depolarization-induced increase in SMMHC/SM &agr;-actin but had no effect on c-fos expression. Conversely, the Ca2+/calmodulin-dependent kinase inhibitor, KN93, prevented depolarization-induced increases in c-fos expression with no effect on SMMHC/SM &agr;-actin. Depolarization increased expression of myocardin, a coactivator of SRF that mediates CArG-dependent transcription of SMC marker gene promoters containing paired CArG cis regulatory elements (SMMHC/SM &agr;-actin). Both nifedipine and Y-27632 prevented the depolarization-induced increase in myocardin expression. Moreover, short interfering RNA (siRNA) specific for myocardin attenuated depolarization-induced SMMHC/SM &agr;-actin transcription. Chromatin immunoprecipitation (ChIP) assays revealed that depolarization increased SRF enrichment of the CArG regions in the SMMHC, SM &agr;-actin, and c-fos promoters in intact chromatin. Whereas Y-27632 decreased basal and depolarization-induced SRF enrichment in the SMMHC/SM &agr;-actin promoter regions, it had no effect of SRF enrichment of c-fos. Taken together, these results provide evidence for a novel mechanism whereby Ca2+ influx via VGCCs stimulates expression of SMC differentiation marker genes through mechanisms that are dependent on ROK, myocardin, and increased binding of SRF to CArG cis regulatory elements.

Strontium Accumulation by Sarcoplasmic Reticulum and Mitochondria in Vascular Smooth Muscle

Electron-opaque deposits of strontium were observed in the sarcoplasmic reticulum and in mitochondria of spontaneously contracting vascular smooth muscles that had been incubated in a strontium-containing solution prior to fixation. The deposits were present in those elements of the sarcoplasmic reticulum that are in close contact with the surface membrane and also in more centrally located portions. In vascular smooth muscle that does not contract spontaneously, similar deposits of strontium were only seen if the muscle was depolarized during or glycerinated before exposure to the strontium-containing solution. Strontium was also deposited in the sarcoplasmic reticulum of the endothelium. It is suggested that translocation of calcium from the sarcoplasmic reticulum that is in close contact with the surface membrane, and now shown to accumulate divalent cations, is responsible for the action potential-triggered contractions of rabbit and guinea pig mesenteric veins. Strontium may also be a suitable marker for identifying sites that accumulate calcium in other types of cells in which translocation of calcium plays a major regulatory function.