Desensitization In Smooth Muscle Research Articles

Binding of smoothelin-like 1 to tropomyosin and calmodulin is mutually exclusive and regulated by phosphorylation

BackgroundThe smoothelin-like 1 protein (SMTNL1) can associate with tropomyosin (Tpm) and calmodulin (CaM), two proteins essential to the smooth muscle contractile process. SMTNL1 is phosphorylated at Ser301 by protein kinase A during calcium desensitization in smooth muscle, yet the effect of SMTNL1 phosphorylation on Tpm- and CaM-binding has yet to be investigated.ResultsUsing pull down studies with Tpm-Sepharose and CaM-Sepharose, we examined the interplay between Tpm binding, CaM binding, phosphorylation of SMTNL1 and calcium concentration. Phosphorylation greatly enhanced the ability of SMTNL1 to associate with Tpm in vitro; surface plasmon resonance yielded a 10-fold enhancement in KD value with phosphorylation. The effect on CaM binding is more complex and varies with the availability of calcium.ConclusionsCombining both CaM and Tpm with SMTNL1 shows that the binding to both is mutually exclusive.

Transient Resensitization Interrupting the Development of Carbachol-induced Desensitization in Smooth Muscle of Guinea-pig Taenia Caeci: Ca<sup>2+</sup>-dependent Termination of Resensitization

It is important to clarify developmental mechanisms of desensitization because of their great significance in regulation of cellular responsiveness. We have found that carbachol-induced desensitization to carbachol develops in three successive phases in the presence of extracellular Ca(2+) in the smooth muscle of guinea pig taenia caeci: fast desensitization within 15 s, transient resensitization reaching a peak at 1 min and the subsequent re-development of desensitization to terminate resensitization for up to 30 min. In contrast, in the absence of extracellular Ca(2+), desensitization develops without resensitization. To further clarify the roles of Ca(2+) in the formation of the transient resensitization phase, we examined the developmental process of carbachol-induced desensitization in the absence of extracellular Ca(2+), following the induction of desensitization by a 15-s treatment with carbachol in the presence of extracellular Ca(2+). Desensitization to carbachol occurred due to pretreatment with 10(-4) M carbachol for 15 s in normal physiological solution, and continued pretreatment with carbachol in Ca(2+)-free solution containing 0.2 mM EGTA induced resensitization followed by the obscure progress of re-desensitization for up to 30 min resulting in a long-lasting phase of resensitization. These results suggest that resensitization is promptly terminated by the Ca(2+)-dependent development of subsequent desensitization for further regulation of cellular responsiveness via G(q) protein-coupled Ca(2+)-mobilizing receptors against sustained stimuli.

Ca2+‐DEPENDENT DIFFERENTIAL DEVELOPMENT OF CARBACHOL‐INDUCED DESENSITIZATION TO RECEPTOR AGONISTS AND HIGH K+ IN GUINEA‐PIG TAENIA CAECI

1. Carbachol (CCh)-induced desensitization to CCh was interrupted by a transient resensitization during its early stage, with concomitant changes at the muscarinic receptor/G-protein level in smooth muscle of guinea-pig taenia caeci. To assess whether such a peculiar desensitizing process may heterologously regulate smooth muscle contraction, we examined the developmental processes of CCh-induced desensitization to histamine and high K(+) and compared it with that to CCh. 2. Under Ca(2+)-containing physiological conditions, treatment with 10(-4) mol/L CCh for 30 min induced heterologous desensitization to histamine and high K(+). The development of desensitization to histamine was interrupted by a transient resensitization at 1 min in a manner similar to that to CCh. In contrast, CCh-induced desensitization to high K(+) reached a peak at 1 min and was followed by a gradual resensitization up to a partial restoration at 30 min. 3. Under Ca(2+)-free conditions containing 0.2 mmol/L EGTA, treatment with 10(-4) mol/L CCh for 30 min failed to induce heterologous desensitization to either histamine or high K(+), whereas the CCh treatment developed homologous desensitization to CCh in a simple time-dependent manner without a resensitization phase. 4. These results suggest that cellular responsiveness to receptor agonists and non-receptor-mediated depolarizing stimulation is differentially regulated by Ca(2+)-dependent heterologous desensitization in smooth muscle.

Modulation of smooth muscle contractility by CHASM, a novel member of the smoothelin family of proteins

Cyclic nucleotides acting through their associated protein kinases, the cGMP- and cAMP-dependent protein kinases, can relax smooth muscles without a change in free intracellular calcium concentration ([Ca 2+] i ), a phenomenon referred to as Ca 2+ desensitization. The molecular mechanisms by which these kinases bring about Ca 2+ desensitization are unknown and an understanding of this phenomenon may lead to better therapies for treating diseases involving defects in the contractile response of smooth muscles such as hypertension, bronchospasm, sexual dysfunction, gastrointestinal disorders and glaucoma. Utilizing a combination of real-time proteomics and smooth muscle physiology, we characterized a distinct subset of protein targets for cGMP-dependent protein kinase in smooth muscle. Among those phosphoproteins identified was calponin homology-associated smooth muscle (CHASM), a novel protein that contains a calponin homology domain and shares sequence similarity with the smoothelin family of smooth muscle specific proteins. Recombinant CHASM was found to evoke relaxation in a concentration dependent manner when added to permeabilized smooth muscle. A co-sedimentation assay with actin demonstrated that CHASM does not possess actin binding activity. Our findings indicate that CHASM is a novel member of the smoothelin protein family that elicits Ca 2+ desensitization in smooth muscle.

Smooth Muscle Phosphatase Is Regulated in Vivo by Exclusion of Phosphorylation of Threonine 696 of MYPT1 by Phosphorylation of Serine 695 in Response to Cyclic Nucleotides

Regulation of smooth muscle myosin phosphatase (SMPP-1M) is thought to be a primary mechanism for explaining Ca(2+) sensitization/desensitization in smooth muscle. Ca(2+) sensitization induced by activation of G protein-coupled receptors acting through RhoA involves phosphorylation of Thr-696 (of the human isoform) of the myosin targeting subunit (MYPT1) of SMPP-1M inhibiting activity. In contrast, agonists that elevate intracellular cGMP and cAMP promote Ca(2+) desensitization in smooth muscle through apparent activation of SMPP-1M. We show that cGMP-dependent protein kinase (PKG)/cAMP-dependent protein kinase (PKA) efficiently phosphorylates MYPT1 in vitro at Ser-692, Ser-695, and Ser-852 (numbering for human isoform). Although phosphorylation of MYPT1 by PKA/PKG has no direct effect on SMPP-1M activity, a primary site of phosphorylation is Ser-695, which is immediately adjacent to the inactivating Thr-696. In vitro, phosphorylation of Ser-695 by PKA/PKG appeared to prevent phosphorylation of Thr-696 by MYPT1K. In ileum smooth muscle, Ser-695 showed a 3-fold increase in phosphorylation in response to 8-bromo-cGMP. Addition of constitutively active recombinant MYPT1K to permeabilized smooth muscles caused phosphorylation of Thr-696 and Ca(2+) sensitization; however, this phosphorylation was blocked by preincubation with 8-bromo-cGMP. These findings suggest a mechanism of Ca(2+) desensitization in smooth muscle that involves mutual exclusion of phosphorylation, whereby phosphorylation of Ser-695 prevents phosphorylation of Thr-696 and therefore inhibition of SMPP-1M.

Acceleration of Myosin Light Chain Dephosphorylation and Relaxation of Smooth Muscle by Telokin: SYNERGISM WITH CYCLIC NUCLEOTIDE-ACTIVATED KINASE

Incorporation of 32P into telokin, a smooth muscle-specific, 17-18-kDa, acidic (pI 4.2-4.4) protein, was increased by forskolin (20 microM) in intact rabbit ileum smooth muscle (ileum) and by 8-bromo-cyclic GMP (100 microM) in alpha-toxin-permeabilized ileum. Native telokin (5-20 microM), purified from turkey gizzard, and recombinant rabbit telokin, expressed in Escherichia coli and purified to >90% purity, induced dose-dependent relaxation, associated with a significant decrease in regulatory myosin light chain phosphorylation, without affecting the rate of thiophosphorylation of regulatory myosin light chain of ileum permeabilized with 0.1% Triton X-100. Endogenous telokin was lost from ileum during prolonged permeabilization (>20 min) with 0.1% Triton X-100, and the time course of loss was correlated with the loss of 8-bromo-cyclic GMP-induced calcium desensitization. Recombinant and native gizzard telokins were phosphorylated, in vitro, by the catalytic subunit of cAMP-dependent protein kinase, cGMP-dependent protein kinase, and p42/44 mitogen-activated protein kinase; the recombinant protein was also phosphorylated by calmodulin-dependent protein kinase II. Exogenous cGMP-dependent protein kinase (0.5 microM) activated by 8-bromo-cyclic GMP (50 microM) phosphorylated recombinant telokin (10 microM) when added concurrently to ileum depleted of its endogenous telokin, and their relaxant effects were mutually potentiated. Forskolin (20 microM) also increased phosphorylation of telokin in intact ileum. We conclude that telokin induces calcium desensitization in smooth muscle by enhancing myosin light chain phosphatase activity, and cGMP- and/or cAMP-dependent phosphorylation of telokin up-regulates its relaxant effect.

O-404 - Changes in muscarinic receptors and their coupling with G proteins during development of Ca2+-independent desensitization in smooth muscle.

O-404 - Changes in muscarinic receptors and their coupling with G proteins during development of Ca2+-independent desensitization in smooth muscle.

P-154 - Functional changes in voltage-dependent Ca channels in heterologous desensitization in smooth muscle: Differential blockade by membrane-stabilizing drugs

P-154 - Functional changes in voltage-dependent Ca channels in heterologous desensitization in smooth muscle: Differential blockade by membrane-stabilizing drugs

Adenosine triphosphate induces a low [Ca2+]i sensitivity of phosphorylation and an unusual form of receptor desensitization in smooth muscle.

The contractile sensitivity of smooth muscle to changes in myoplasmic [Ca2+] is dependent on the form of stimulation. Both myosin phosphorylation and force are less sensitive to increases in [Ca2+]i derived from Ca2+ entry through L-type Ca2+ channels than to increases in [Ca2+] induced by agents which release internal Ca2+ stores. We hypothesized that activation of receptor-operated channels should produce a [Ca2+]i sensitivity similar to that induced by opening L channels. Aequorin-estimated myoplasmic [Ca2+] and myosin light chain phosphorylation were measured in swine carotid media tissues stimulated with ATP, an activator of the only known receptor-operated cation channel in smooth muscle. ATP, via activation of a P2x purinergic receptor, induced large, transient increases in [Ca2+]i, yet only small transient elevations in phosphorylation or force. Rapid desensitization to ATP was partially, but not completely, caused by hydrolysis of ATP into adenosine since 1) alpha-beta-methylene ATP (a poorly hydrolyzable analog of ATP) produced larger, yet still transient increases in [Ca2+]i, phosphorylation, and force; 2) BW A1433U, a P1 (adenosine) receptor antagonist, enhanced ATP-induced contractions; and 3) ATP, but not alpha-beta-methylene ATP increased bath [adenosine]. The [Ca2+]i sensitivity of phosphorylation during P2x receptor activation was similar to that observed with KCl-depolarization-induced opening of L channels, supporting the hypothesis that transplasmalemmal Ca2+ influx produces less phosphorylation and force than mobilization of intracellular Ca2+ stores. Cumulative additions of higher alpha-beta-methylene ATP concentrations induced repeated transient contractions, indicative of an unusual form of receptor desensitization which could be explained if the affinity of the P2x receptor for ATP, but not the receptor number were rapidly reduced.

The effects of nucleotides on the response of the isolated guinea pig urinary bladder to nonadrenergic, noncholinergic nerve stimulation.

The ability of adenosine 5'-triphosphate (ATP) to suppress the contractile response of guinea pig urinary bladder to nonadrenergic, noncholinergic nerve stimulation was investigated. Strips of guinea pig urinary bladder were incubated for 1 h at 37 degrees C in a modified Krebs solution containing atropine (2 X 10(-7) M), guanethidine (1 X 10(-6) M), and indomethacin (5.5 X 10(-6) M). Four successive administrations of ATP, guanosine, 5'-triphosphate (GTP), and cytidine 5'-triphosphate (CTP), each at 5 X 10(-4) M, in the absence of washout resulted in complete desensitization of the tissue to the contractile effect of the nucleotide. Strips desensitized to GTP or CTP were also nonresponsive to ATP. The response of the strips to electrical stimulation (100 V, 0.1-ms pulse, 5-s train at 3 Hz) was markedly reduced following desensitization with ATP but only slightly with GTP or CTP. Under similar conditions, adenosine 5'-monophosphatase (AMP) or diphosphate (ADP) or adenosine (A) also reduced the response of the strips to intramural nerve stimulation, but guanosine 5'-monophosphate (GMP) or diphosphate (GDP), guanosine (G), cytidine 5'-monophosphate (CMP) or diphosphate (CDP), or cytidine (C) did not. The present data suggest that desensitization of smooth muscle to exogenous ATP may be predominantly the result of its conversion to metabolic products rather than inactivation of "purinergic" receptor per se.