Endothelin increases myofilament Ca2+ sensitivity in alpha-toxin-permeabilized rabbit mesenteric artery.

This study was designed to investigate the mechanism of endothelin-1 (ET-1) contractions in Staphylococcus alpha-toxin-permeabilized vascular smooth muscle. Rabbit small mesenteric arteries permeabilized with alpha-toxin were mounted for isometric or isotonic force recording or were processed for determination of myosin light chain (MLC) phosphorylation levels. Addition of 100 nM ET-1 plus 10 microM GTP significantly enhanced myofilament Ca2+ sensitivity as compared with the addition of Ca2+ alone (EC50, 0.47 microM Ca2+ for Ca2+ alone and 0.13 microM Ca2+ for ET-1 plus (GTP). This enhanced sensitivity was reversed by GDP beta S. ET-1-induced contractions were relaxed at a constant [Ca2+] by the addition of 30 microM cAMP or cGMP, demonstrating a direct effect of the cyclic nucleotides on contractile regulation. Inhibition of protein kinase C activity by 100 nM staurosporine relaxed ET-1 plus GTP-induced contractions, and pretreatment with 40 microM chelerythrine inhibited the ET-1 plus GTP increase in force. At 0.32 microM Ca2+, steady-state levels of shortening velocity were not increased by ET-1 plus GTP, although steady-state levels of MLC phosphorylation were significantly enhanced. The ET-1-induced increase in MLC phosphorylation was not altered by changes in [Ca2+], whereas the shortening velocity was Ca2+ dependent, suggesting that the increase MLC phosphorylation level may be the result of protein kinase C, rather than MLC kinase, activation. These results are consistent with the hypothesis that ET-1 increases myofilament Ca2+ sensitivity by a G protein-dependent pathway and subsequent activation of protein kinase C.(ABSTRACT TRUNCATED AT 250 WORDS)     

G protein-mediated inhibition of myosin light-chain phosphatase in vascular smooth muscle.

The mechanism of G protein-mediated sensitization of the contractile apparatus of smooth muscle to Ca2+ was studied in receptor-coupled alpha-toxin-permeabilized rabbit portal vein smooth muscle. To test the hypothesis that Ca2+ sensitization is due to inhibition of myosin light-chain (MLC) phosphatase activity, we measured the effect of guanosine 5'-[gamma-thio]triphosphate and phenylephrine on the rate of MLC dephosphorylation in muscles preactivated with Ca2+ and incubated in Ca(2+)- and ATP-free solution containing 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9) to block MLC kinase activity. Guanosine 5'-[gamma-thio]triphosphate alone (300 microM) or in combination (3 microM) with phenylephrine decreased the rates of relaxation and dephosphorylation of MLC to about half of control values; this inhibition is sufficient to account for maximal G protein-mediated Ca2+ sensitization of MLC phosphorylation. The rate of thiophosphorylation of MLC with adenosine 5'-[gamma-thio]-triphosphate was not affected by guanosine 5'-[gamma-thio]triphosphate. We suggest that inhibition of protein phosphatase(s) by G protein(s) may have important regulatory functions.     

Calcium dependence of neurotensin stimulation of circular colonic muscle of the rabbit

The effect of neurotensin on smooth muscle contraction was compared in strips from rabbit proximal and distal circular colonic muscle. The effective dose for neurotensin stimulation that caused a 50% response in both tissues was similar (1.3 X 10(-10) M). The maximal isometric stress, however, was greater in the distal colon than in the proximal colon (p less than 0.01). Neurotensin stimulation of both proximal and distal colon was unaffected by tetrodotoxin, phentolamine, propranolol, naloxone, or atropine. Neurotensin-stimulated contraction was inhibited by "Ca2+-free" (pCa = 5.1) or La3+ buffer. Verapamil (10(-6) M) or nitroprusside (10(-4) M) decreased neurotensin stimulation of proximal and distal colon by approximately 40% (p less than 0.05). Removal of Ca2+ from the buffer inhibited stimulation of muscle contraction by high extracellular potassium [( K+]o) more than bethanechol stimulation (p less than 0.01). La3+ (1 mM) inhibited the contraction stimulated by bethanechol or increased [K+]o. Although verapamil inhibited contraction by bethanechol and increased [K+]o by approximately 50%, nitroprusside had no effect on the contraction mediated by these stimulants. 8-Bromo-guanosine 3',5'-cyclic monophosphate (cGMP) inhibited neurotensin, but not [K+]o or bethanechol-stimulated contraction. These data suggest (a) neurotensin stimulated colonic contractions at a concentration that is potentially physiologic, (b) neurotensin stimulated colonic smooth muscle directly without neural mediation, (c) neurotensin stimulation of colonic muscle is controlled by [Ca2+]o and [cGMP]i.     

Multiple signaling pathways control stimulus-secretion coupling in rat peritoneal mast cells.

Fura-2 and membrane capacitance measurements were performed to investigate intracellular Ca2+ concentration [( Ca2+]i) and secretory responses of rat peritoneal mast cells following secretagogue stimulation. Compound 48/80 and internally applied guanosine 5'-[gamma-thio]triphosphate (GTP[gamma-S]) induced transient rises in [Ca2+]i and caused membrane capacitance increases as secretion occurred. The 48/80-induced Ca2+ transients and secretory responses were blocked by guanosine 5'-[beta-thio]diphosphate and neomycin, indicating that inositolphospholipid breakdown mediated by guanine nucleotide-binding regulatory protein (G protein) plays an important role in stimulus-secretion coupling. However, pertussis toxin did not block Ca2+ transients induced by 48/80 or GTP[gamma-S], whereas secretory responses were either abolished (48/80) or developed only after a considerable delay (GTP[gamma-S]). Similar effects were obtained by perfusing cells with cAMP: (i) Ca2+ transients following stimulation with 48/80 remained unaffected by cAMP, but secretory responses were abolished; (ii) GTP[gamma-S] induced normal Ca2+ transients and degranulation in the presence of cAMP. Pretreatment of mast cells with phorbol 12-myristate 13-acetate (PMA) abolished 48/80- and GTP[gamma-S]-induced Ca2+ transients (but not inositol trisphosphate-induced Ca2+ transients), whereas secretion still occurred. At the same time, the Ca2+ requirement for secretion was reduced by PMA. These results indicate that secretion in mast cells is under control of an as yet unidentified signaling pathway that involves a G protein. This pathway is distinct from inositolphospholipid turnover and may provide the triggering mechanism for secretion, whereas the inositolphospholipid pathway serves to increase [Ca2+]i and renders the secretory process more sensitive to [Ca2+]i by activating protein kinase C. Persistent activation of protein kinase C through phorbol ester imposes negative feedback control on the inositolphospholipid pathway, whereas cAMP may inhibit the unidentified signaling pathway.     

Sphingosylphosphorylcholine is a novel messenger for Rho-kinase-mediated Ca2+ sensitization in the bovine cerebral artery: unimportant role for protein kinase C

Although recent investigations have suggested that a Rho-kinase-mediated Ca2+ sensitization of vascular smooth muscle contraction plays a critical role in the pathogenesis of cerebral and coronary vasospasm, the upstream of this signal transduction has not been elucidated. In addition, the involvement of protein kinase C (PKC) may also be related to cerebral vasospasm. We recently reported that sphingosylphosphorylcholine (SPC), a sphingolipid, induces Rho-kinase-mediated Ca2+ sensitization in pig coronary arteries. The purpose of this present study was to examine the possible mediation of SPC in Ca2+ sensitization of the bovine middle cerebral artery (MCA) and the relation to signal transduction pathways mediated by Rho-kinase and PKC. In intact MCA, SPC induced a concentration-dependent (EC50=3.0 micromol/L) contraction, without [Ca2+]i elevation. In membrane-permeabilized MCA, SPC induced Ca2+ sensitization even in the absence of added GTP, which is required for activation of G-proteins coupled to membrane receptors. The SPC-induced Ca2+ sensitization was blocked by a Rho-kinase inhibitor (Y-27632) and a dominant-negative Rho-kinase, but not by a pseudosubstrate peptide for conventional PKC, which abolished the Ca2+-independent contraction induced by phorbol ester. In contrast, phorbol ester-induced Ca2+ sensitization was resistant to a Rho-kinase inhibitor and a dominant-negative Rho-kinase. In primary cultured vascular smooth muscle cells, SPC induced the translocation of cytosolic Rho-kinase to the cell membrane. We propose that SPC is a novel messenger for Rho-kinase-mediated Ca2+ sensitization of cerebral arterial smooth muscle and, therefore, may play a pivotal role in the pathogenesis of abnormal contraction of the cerebral artery such as vasospasm. The SPC/Rho-kinase pathway functions independently of the PKC pathway.     

Interaction between src family kinases and rho-kinase in agonist-induced Ca2+-sensitization of rat pulmonary artery

AIMS: We investigated the role of src family kinases (srcFK) in agonist-mediated Ca2+-sensitization in pulmonary artery and whether this involves interaction with the rho/rho-kinase pathway. METHODS AND RESULTS: Intra-pulmonary arteries (IPAs) and cultured pulmonary artery smooth muscle cells (PASMC) were obtained from rat. Expression of srcFK was determined at the mRNA and protein levels. Ca2+-sensitization was induced by prostaglandin F(2 alpha) (PGF(2 alpha)) in alpha-toxin-permeabilized IPAs. Phosphorylation of the regulatory subunit of myosin phosphatase (MYPT-1) and of myosin light-chain-20 (MLC20) and translocation of rho-kinase in response to PGF(2 alpha) were also determined. Nine srcFK were expressed at the mRNA level, including src, fyn, and yes, and PGF(2 alpha) enhanced phosphorylation of three srcFK proteins at tyr-416. In alpha-toxin-permeabilized IPAs, PGF(2 alpha) enhanced the Ca2+-induced contraction (pCa 6.9) approximately three-fold. This enhancement was inhibited by the srcFK blockers SU6656 and PP2 and by the rho-kinase inhibitor Y27632. Y27632, but not SU6656 or PP2, also inhibited the underlying pCa 6.9 contraction. PGF(2 alpha) enhanced phosphorylation of MYPT-1 at thr-697 and thr-855 and of MLC20 at ser-19. This enhancement, but not the underlying basal phosphorylation, was inhibited by SU6656. Y27632 suppressed both basal and PGF(2 alpha)-mediated phosphorylation. The effects of SU6656 and Y27632, on both contraction and MYPT-1 and MLC20 phosphorylation, were not additive. PGF(2 alpha) triggered translocation of rho-kinase in PASMC, and this was inhibited by SU6656. CONCLUSIONS: srcFK are activated by PGF(2 alpha) in the rat pulmonary artery and may contribute to Ca2+-sensitization and contraction via rho-kinase translocation and phosphorylation of MYPT-1.     

Control of intracellular calcium redistribution by guanine nucleotides and inositol 1,4,5-trisphosphate in permeabilized GH4C1 cells

In GH4C1 rat pituitary cells, a GTP-binding protein appears to be involved in signal transduction between the TRH receptor and phospholipase C. In certain other cell types, another role for GTP has been reported, namely regulation of Ca2+ translocation from one intracellular pool to another. Using digitonin-permeabilized GH4C1 cells, we have investigated whether an analogous process occurs in pituitary cells. In permeabilized GH4C1 cells, TRH, inositol 1,4,5-trisphosphate (IP3), and nonhydrolyzable GTP analogs guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and 5'-guanylyl imidodiphosphate each increased free Ca2+ concentration [( Ca2+]). Unlike several other systems, GTP did not increase [Ca2+]. Guanosine 5'-O-(2-thiodiphosphate) inhibited Ca2+ release induced by both TRH and GTP gamma S. Heparin abolished IP3-induced Ca2+ release but did not prevent Ca2+ release induced by TRH or GTP gamma S, suggesting a mechanism for their actions that did not depend solely on IP3 production. Neomycin inhibited GTP gamma S-induced Ca2+ release, but it did not prevent TRH- or IP3-induced Ca2+ release. In the absence of ATP, GTP gamma S did not elevate [Ca2+], although TRH and IP3 did, suggesting that ATP-dependent sequestration of Ca2+ was necessary for the action of GTP gamma S in this system, but not for TRH and IP3. Repeated additions of IP3 resulted in an attenuation of the response to IP3- GTP gamma S, which itself increased [Ca2+] after IP3 attenuation, restored the attenuated Ca2+ response to IP3. We conclude that, in permeabilized GH4C1 cells, GTP gamma S as well as TRH cause intracellular Ca2+ release; however, their mechanisms of action are, at least in part, distinct. Furthermore, the IP3-depletable Ca2+ pool can be refilled from a GTP gamma S-sensitive compartment via Ca2+ transport through the cytosol.     

Phospholipase A2 and phospholipase C are activated by distinct GTP-binding proteins in response to alpha 1-adrenergic stimulation in FRTL5 thyroid cells.

In FRTL5 rat thyroid cells, norepinephrine, by interacting with alpha 1-adrenergic receptors, stimulates inositol phosphate formation, through activation of phospholipase C, and arachidonic acid release. Recent studies have shown that GTP-binding proteins couple several types of receptors to phospholipase C activation. The present study was undertaken to determine whether GTP-binding proteins couple alpha 1-adrenergic receptors to stimulation of phospholipase C activity and arachidonic acid release. When introduced into permeabilized FRTL5 cells, guanosine 5'-[gamma-thio]triphosphate (GTP[gamma-S]), which activates many GTP-binding proteins, stimulated inositol phosphate formation and arachidonic acid release. Neomycin inhibited GTP[gamma-S]-stimulated inositol phosphate formation but was without effect on GTP[gamma-S]-stimulated arachidonic acid release, suggesting that separate GTP-binding proteins mediate each process. In addition, pertussis toxin inhibited norepinephrine-stimulated arachidonic acid release but not norepinephrine-stimulated inositol phosphate formation. Norepinephrine-stimulated arachidonic acid release but not inositol phosphate formation was also inhibited by decreased extracellular calcium and by TMB-8, suggesting a role for a phospholipase A2. To confirm that arachidonic acid was released by a phospholipase A2, FRTL5 membranes were incubated with 1-acyl-2-[3H]arachidonoyl-sn-glycero-3-phosphocholine. GTP[gamma-S] slightly stimulated arachidonic acid release, whereas norepinephrine acted synergistically with GTP[gamma-S] to stimulate arachidonic acid release. The results show that phospholipase C and phospholipase A2 are activated by alpha 1-adrenergic agonists. Both phospholipases are coupled to the receptor by GTP-binding proteins. That coupled to phospholipase A2 is pertussis toxin-sensitive, whereas that coupled to phospholipase C is pertussis toxin-insensitive.     

Smooth muscle of telokin-deficient mice exhibits increased sensitivity to Ca2+ and decreased cGMP-induced relaxation

Cyclic nucleotides can relax smooth muscle without a change in [Ca2+]i, a phenomenon termed Ca2+ desensitization, contributing to vasodilation, gastrointestinal motility, and airway resistance. The physiological importance of telokin, a 17-kDa smooth muscle-specific protein and target for cyclic nucleotide-induced Ca2+ desensitization, was determined in telokin null mice bred to a congenic background. Telokin null ileal smooth muscle homogenates compared to wild type exhibited an approximately 30% decrease in myosin light-chain phosphatase (MLCP) activity, which was reflected in a significant leftward shift (up to 2-fold at pCa 6.3) of the Ca2+ force relationship accompanied by an increase in myosin light-chain phosphorylation. No difference in the Ca2+ force relationship occurred in telokin WT and knockout (KO) aortas, presumably reflecting the normally approximately 5-fold lower telokin content in aorta vs. ileum smooth muscle. Ca2+ desensitization of contractile force by 8-Br-cGMP was attenuated by 50% in telokin KO intestinal smooth muscle. The rate of force relaxation reflecting MLCP activity, in the presence of 50 microM 8-Br-cGMP, was also significantly slowed in telokin KO vs. WT ileum and was rescued by recombinant telokin. Normal thick filaments in telokin KO smooth muscles indicate that telokin is not required for filament formation or stability. Results indicate that a primary role of telokin is to modulate force through increasing MLCP activity and that this effect is further potentiated through phosphorylation by cGMP in telokin-rich smooth tissues.     

Bacterial ADP-ribosyltransferase with a substrate specificity of the rho protein disassembles the Golgi apparatus in Vero cells and mimics the action of brefeldin A.

Epidermal-cell differentiation inhibitor (EDIN) is an exoenzyme produced by Staphylococcus aureus that catalyzes the ADP-ribosylation of rho proteins, members of the small GTP-binding protein family. In this study we demonstrate that EDIN induces a rapid morphological change in the Golgi structure of monkey kidney Vero cells that is similar to the changes elicited by brefeldin A (BFA). Treatment of Vero cells with EDIN resulted in a rapid disappearance of N-7-(4-nitrobenzo-2-oxa-1,3-diazole)-6-aminocaproylsphingosine, a 110-kDa protein (beta-COP, coat protein), and mannosidase II from the Golgi structure. Lower doses of EDIN and BFA had a synergistic effect on the redistribution of the Golgi markers. The similarities in the effects of EDIN and BFA in Vero cells also include the EDIN- or BFA-mediated protection of Vero cells from ricin cytotoxicity and prevention of the effects of EDIN or BFA on the distribution of Golgi markers by the pretreatment of Vero cells with guanosine 5'-[gamma-thio]triphosphate or forskolin. Incubation of a Vero-cell homogenate with [32P]NAD+ and EDIN in vitro resulted in the appearance of a labeled band with an apparent molecular mass of 22 kDa. The morphological change of the Golgi structure induced by EDIN was inhibited by nicotinamide, an inhibitor of EDIN-catalyzed ADP-ribosylation. Thus these data suggest that a rho protein is involved in the membrane trafficking between the Golgi and the endoplasmic reticulum of Vero cells and that this rho protein may be a target shared by EDIN and BFA.     

Regulation of vascular tone by the modulation of Ca2+ sensitivity

The contractile tone of the vascular smooth muscle plays an important role on the regulation of the blood pressure as well as the local perfusion of the important organs such as the heart and brain. The importance of the Ca(2+) sensitivity in the regulation of the vascular tone has been established by the development of the simultaneous measurements of intracellular Ca(2+) concentration ( [Ca(2+)](i) ) and tension as well as that of the receptor coupled permeabilized preparation in the late 1980s. Recently, the mechanisms underlying the regulation of Ca(2+) sensitivity have been revealed. The increase in the Ca(2+) sensitivity involves the myosin phosphatase (MLCP) inhibition mediated by rhoA-rho kinase system and PKC-CPI system. The decrease in the Ca(2+) sensitivity involves the PKA-mediated inhibition of myosin light chain kinase, the PKG-mediated activation of MLCP, and PKA- or PKG-mediated inactivation of rhoA. In this article, the regulation of the Ca(2+) sensitivity of the contractile apparatus of the vascular smooth muscle will be briefly reviewed.     

Ca2+-dependent rapid Ca2+ sensitization of contraction in arterial smooth muscle

Ca(2+) ion is a universal intracellular messenger that regulates numerous biological functions. In smooth muscle, Ca(2+) with calmodulin activates myosin light chain (MLC) kinase to initiate a rapid MLC phosphorylation and contraction. To test the hypothesis that regulation of MLC phosphatase is involved in the rapid development of MLC phosphorylation and contraction during Ca(2+) transient, we compared Ca(2+) signal, MLC phosphorylation, and 2 modes of inhibition of MLC phosphatase, phosphorylation of CPI-17 Thr38 and MYPT1 Thr853, during alpha(1) agonist-induced contraction with/without various inhibitors in intact rabbit femoral artery. Phenylephrine rapidly induced CPI-17 phosphorylation from a negligible amount to a peak value of 0.38+/-0.04 mol of Pi/mol within 7 seconds following stimulation, similar to the rapid time course of Ca(2+) rise and MLC phosphorylation. This rapid CPI-17 phosphorylation was dramatically inhibited by either blocking Ca(2+) release from the sarcoplasmic reticulum or by pretreatment with protein kinase C inhibitors, suggesting an involvement of Ca(2+)-dependent protein kinase C. This was followed by a slow Ca(2+)-independent and Rho-kinase/protein kinase C-dependent phosphorylation of CPI-17. In contrast, MYPT1 phosphorylation had only a slow component that increased from 0.29+/-0.09 at rest to the peak of 0.68+/-0.14 mol of Pi/mol at 1 minute, similar to the time course of contraction. Thus, there are 2 components of the Ca(2+) sensitization through inhibition of MLC phosphatase. Our results support the hypothesis that the initial rapid Ca(2+) rise induces a rapid inhibition of MLC phosphatase coincident with the Ca(2+)-induced MLC kinase activation to synergistically initiate a rapid MLC phosphorylation and contraction in arteries with abundant CPI-17 content.     

Inositol trisphosphate-induced calcium release and contraction in vascular smooth muscle.

Inositol 1,4,5-trisphosphate (InsP3) caused Ca release and tension development in rabbit main pulmonary artery smooth muscle permeabilized with saponin or digitonin. Both of these responses to single additions of InsP3 (0.5-30 microM) were repeatable and occurred in the presence of 0.0-1.9 mM free Mg2+. Sustained contractions were induced by InsP3. The amount of Ca released by InsP3, measured with a Ca2+-selective electrode, was also estimated to be sufficient to stimulate contraction in intact smooth muscle. Ca release was not influenced by inhibitors of mitochondrial oxidative phosphorylation. The uptake of Ca2+ from the medium into the InsP3-sensitive pool was ATP-dependent. The present results support the hypothesis that, in smooth muscle, InsP3 is the messenger, or one of the messengers, involved in transmitter-induced (pharmacomechanical) Ca release from the sarcoplasmic reticulum, which is the intracellular Ca store identified previously as the source of Ca released by norepinephrine in main pulmonary artery.     

Regulation of phospholipid hydrolysis in streptolysin-O-permeabilized rat pancreatic acini.

To investigate the mechanism of phospholipid hydrolysis in pancreatic acinar cells, the effects of Ca2+, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and cholecystokinin (CCK) on both polyphosphoinositide (PI) and phosphatidylcholine (PC) hydrolysis were studied in rat pancreatic acini permeabilized with the bacterial toxin, streptolysin-O. When acini were prelabeled with myo-[3H]inositol, permeabilized, and then incubated with various concentrations of free Ca2+ for 15 min, Ca2+ stimulated [3H]inositol phosphate release at a concentration of 100 nM and was maximally effective at 100 microM. Both GTP gamma S and CCK enhanced Ca(2+)-induced [3H]inositol phosphate release, although these agents had no effect in the absence of Ca2+. At a physiological concentration of Ca2+ (100 nM), CCK stimulated [3H]inositol phosphate release which was further enhanced by GTP gamma S. When acini were similarly prelabeled with [3H]choline before permeabilization, [3H]choline phosphate release was also stimulated by free Ca2+ over the concentration range from 100 nM to 10 microM. In contrast to PI hydrolysis, however, neither GTP gamma S, CCK, or GTP gamma S plus CCK had an additional effect on [3H]choline phosphate release stimulated by 100 nM-100 microM free Ca2+. Furthermore, Ca(2+)-induced [3H]choline phosphate release appeared to be due to the redistribution from cell to the medium rather than to an increase in choline phosphate production. Therefore, choline phosphate release following prelabeling with [3H]choline is not useful as an indicator of PC hydrolysis in permeabilized acini.     

Regulation of calcium current by intracellular calcium in smooth muscle cells of rabbit portal vein

Effects of concentrations of intracellular calcium, [Ca2+]i, on the voltage-dependent Ca2+ current (ICa) recorded from dispersed single smooth muscle cells of the rabbit portal vein were studied, using a whole cell voltage clamp method combined with an intracellular perfusion technique. Outward currents were minimized by replacement of Cs+ -rich solution in the pipette and 20 mM tetraethylammonium in the bath. The ICa was evoked by command pulses of above -30 mV, and the maximum amplitude was obtained at about 0 mV. This ICa was dose dependently inhibited by increases in the [Ca2+]i above 30 nM. The Kd value of the [Ca2+]i required to inhibit the ICa was about 100 nM. The Ba2+ current was also inhibited by increases in the [Ca2+]i. Conversely, perfusion of Ba2+ into the cell up to 100 microM did not suppress the ICa. Changes in the [Ca2+]i did not modify the steady-state inactivation curve. The inhibition of the ICa evoked by the test pulse is most prominent when the preceding influx of Ca2+ during the conditioning pulse was large, as estimated using a double pulse protocol. This inhibition was proportionally reduced by increases in the concentration of the Ca2+ chelator, ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). Therefore, the Ca2+ -dependent inactivation of the Ca2+ channel may contribute toward regulating [Ca2+]i in smooth muscle cells of the rabbit portal vein.     

Relationship between force and Ca2+ concentration in smooth muscle as revealed by measurements on single cells.

The role of Ca2+ in regulating smooth muscle contraction was investigated by measuring isometric force and [Ca2+] simultaneously in individual single smooth-muscle cells. [Ca2+] was measured with fura-2 and a high time-resolution dual-wavelength digital microfluorimeter, and force was measured with an ultrasensitive force transducer attached to a probe around which was tied one end of the cell. Both [Ca2+] and force increase after maximal electrical stimulus, with [Ca2+] increasing considerably before the first detectable increase in force. Force development exhibited maximal sensitivity to [Ca2+] between 150 and 500 nM Ca2+. This Ca2+ sensitivity can account for the fact that many physiological stimuli produce full contraction even though such stimuli only increase Ca2+ to 600-800 nM. When Ca2+ was induced to increase rapidly, the relation between [Ca2+] and force exhibited hysteresis. During the onset of contraction, force at a given [Ca2+] was lower than during the muscle's return to rest, thus suggesting the existence of a slow step(s) linking Ca2+ and force development in smooth muscle. The direction of this hysteresis reversed during contractions in which Ca2+ increased slowly, suggesting that the contractile process becomes desensitized to [Ca2+] with time. These relations between calcium and force in intact single smooth-muscle cells differ in many respects from the relation found previously in chemically permeabilized multicellular preparations of smooth muscle.     

G protein-dependent presynaptic inhibition mediated by AMPA receptors at the calyx of Held

The alpha-amino-3-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) is an ionotropic receptor mediating excitatory synaptic transmission, but it can also interact with intracellular messengers. Here we report that, at the calyx of Held in the rat auditory brainstem, activation of AMPARs induced inward currents in the nerve terminal and inhibited presynaptic Ca2+ currents (I(pCa)), thereby attenuating glutamatergic synaptic transmission. The AMPAR-mediated I(pCa) inhibition was disinhibited by a strong depolarizing pulse and occluded by the nonhydrolyzable GTP analog GTPgammaS loaded into the terminal. We conclude that functional AMPARs are expressed at the calyx of Held nerve terminal and that their activation inhibits voltage-gated Ca2+ channels by an interaction with heterotrimeric GTP-binding proteins (G proteins). Thus, at a central glutamatergic synapse, presynaptic AMPARs have a metabotropic nature and regulate transmitter release by means of G proteins.     

Calcium ion-insensitive contraction of glycerinated porcine cardiac muscle fibers by Mg-inosine triphosphate. ITP as a tool to dissociate the contraction mechanism from the regulatory mechanism

The contraction of cardiac muscle that has been treated with glycerol requires Ca2+ (pCa 8-5), when MgATP is used as a substrate. In contrast, this preparation contracts, even in the absence of Ca2+ (pCa 8-10), when ATP is replaced by ITP. Ca2+ dependency was not observed after increasing free Ca2+ concentrations from pCa 8.0 to 5.0, or after increasing MgITP concentration from 5 to 80 mM. On the other hand, rabbit skeletal muscle fiber treated by the same method as cardiac muscle demonstrates Ca2+ dependency in the presence of both MgITP and MgATP, although this Ca2+ regulation is less in the presence of MgITP. Loss of Ca2+ dependency was confirmed by the finding that, in contrast to ATPase, the ITPase activity of cardiac myofibrils was not dependent on Ca2+ concentrations. Furthermore, the very fast tension responses (quick phases) following quick stretch and quick release were missing in MgITP, and the contractions were similar to rigor. These were not rigor however, because phosphate liberation from ITP continued, and muscle shortening occurred in MgITP. These findings suggest that MgITP dissociates the contraction mechanism from the regulatory mechanism, modulating the regulatory properties of cardiac muscle fiber.     

Stimulation of phospholipase A2 activity in bovine rod outer segments by the beta gamma subunits of transducin and its inhibition by the alpha subunit.

In the rod outer segments (ROS) of bovine retina, light activation of phospholipase A2 has been shown to occur by a transducin-dependent mechanism. In this report, the transducin-mediated stimulation of phospholipase A2 is shown to require dissociation of the alpha beta gamma heterotrimer. Addition of transducin to dark-adapted transducin-poor ROS stimulated phospholipase A2 activity only with coincident exposure to white light or, in the dark, with addition of the hydrolysis-resistant GTP analog, guanosine 5'-[gamma-thio]triphosphate (GTP[gamma-S]). Both light and GTP[gamma-S] induced dissociation of the transducin subunits and led to severalfold increases in the phospholipase A2 activity of transducin-rich, but not transducin-poor, ROS. In contrast, pertussis toxin treatment of transducin, which stabilizes the associated state of this G protein, prevented the stimulation of phospholipase A2 by exogenous transducin in the presence of light. Addition of purified transducin subunits to dark-adapted transducin-poor ROS revealed that phospholipase A2 stimulation occurred by action of the beta gamma subunits. This is in contrast to the transducin-mediated increase in cGMP phosphodiesterase activity, where activation occurs by action of the alpha subunit. The alpha subunit, which itself slightly stimulated phospholipase A2 activity, inhibited the beta gamma-induced stimulation of phospholipase A2. This inhibition appears to be the result of subunit reassociation since addition of GTP[gamma-S] abolished the inhibitory effect of the alpha subunit on the beta gamma-induced increase in phospholipase A2, while pertussis toxin treatment of the subunits further inhibited phospholipase A2 activity. Modulation of phospholipase A2 activity by the transducin subunit is, therefore, a mode of action for these subunits in signal transduction.     

Urocortin-induced decrease in Ca2+ sensitivity of contraction in mouse tail arteries is attributable to cAMP-dependent dephosphorylation of MYPT1 and activation of myosin light chain phosphatase

Urocortin, a vasodilatory peptide related to corticotropin-releasing factor, may be an endogenous regulator of blood pressure. In vitro, rat tail arteries are relaxed by urocortin by a cAMP-mediated decrease in myofilament Ca2+ sensitivity through a still unclear mechanism. Here we show that contraction of intact mouse tail arteries induced with 42 mmol/L KCl or 0.5 micromol/L noradrenaline was associated with a approximately 2-fold increase in the phosphorylation of the regulatory subunit of myosin phosphatase (SMPP-1M), MYPT1, at Thr696, which was reversed in arteries relaxed with urocortin. Submaximally (pCa 6.1) contracted mouse tail arteries permeabilized with alpha-toxin were relaxed with urocortin by 39+/-3% at constant [Ca2+], which was associated with a decrease in myosin light chain (MLC20Ser19), MYPT1Thr696, and MYPT1Thr850 phosphorylation by 60%, 28%, and 52%, respectively. The Rho-associated kinase (ROK) inhibitor Y-27632 decreased MYPT1 phosphorylation by a similar extent. Inhibition of PP-2A with 3 nmol/L okadaic acid had no effect on MYPT1 phosphorylation, whereas inhibition of PP-1 with 3 micromol/L okadaic acid prevented dephosphorylation. Urocortin increased the rate of dephosphorylation of MLC20Ser19 approximately 2.2-fold but had no effect on the rate of contraction under conditions of, respectively, inhibited kinase and phosphatase activities. The effect of urocortin on MLC20Ser19 and MYPT1 phosphorylation was blocked by Rp-8-CPT-cAMPS and mimicked by Sp-5,6-DCl-cBIMPS. In summary, these results provide evidence that Ca(2+)-independent relaxation by urocortin can be attributed to a cAMP-mediated increased activity of SMPP-1M which at least in part is attributable to a decrease in the inhibitory phosphorylation of MYPT1.