Phosphorylation of the small heat shock-related protein, HSP20, in vascular smooth muscles is associated with changes in the macromolecular associations of HSP20

Cyclic nucleotide-dependent vasorelaxation is associated with increases in the phosphorylation of a small heat shock-related protein, HSP20. We hypothesized that phosphorylation of HSP20 in vascular smooth muscles is associated with alterations in the macromolecular associations of HSP20. Treatment of bovine carotid artery smooth muscles with the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, and the adenylate cyclase activator, forskolin, led to increases in the phosphorylation of HSP20 and dissociation of macromolecular aggregates of HSP20. However, 3-isobutyl-1-methylxanthine and forskolin treatment of a muscle that is uniquely refractory to cyclic nucleotide-dependent vasorelaxation, human umbilical artery smooth muscle, did not result in increases in the phosphorylation of HSP20 or to dissociation of macromolecular aggregates. HSP20 can be phosphorylated in vitro by the catalytic subunit of cAMP-dependent protein kinase (PKA) in both carotid and umbilical arteries and this phosphorylation of HSP20 is associated with dissociation of macromolecular aggregates of HSP20. Activation of cyclic nucleotide-dependent signaling pathways does not lead to changes in the macromolecular associations of another small heat shock protein, HSP27. Interestingly, the myosin light chains (MLC20) are in similar fractions as the HSP20, and phosphorylation of HSP20 is associated with changes in the macromolecular associations of MLC20. These data suggest that increases in the phosphorylation of HSP20 are associated with changes in the macromolecular associations of HSP20. HSP20 may regulate vasorelaxation through a direct interaction with specific contractile regulatory proteins.     

PI3-kinase/Akt modulates vascular smooth muscle tone via cAMP signaling pathways.

Phosphatidylinositol 3-kinase (PI3-kinase) activates protein kinase B (also known as Akt), which phosphorylates and activates a cyclic nucleotide phosphodiesterase 3B. Increases in cyclic nucleotide concentrations inhibit agonist-induced contraction of vascular smooth muscle. Thus we hypothesized that the PI3-kinase/Akt pathway may regulate vascular smooth muscle tone. In unstimulated, intact bovine carotid artery smooth muscle, the basal phosphorylation of Akt was higher than that in cultured smooth muscle cells. The phosphorylation of Akt decreases in a time-dependent manner when incubated with the PI3-kinase inhibitor, LY-294002. Agonist (serotonin)-, phorbol ester (phorbol 12,13-dibutyrate; PDBu)-, and depolarization (KCl)-induced contractions of vascular smooth muscles were all inhibited in a dose-dependent fashion by LY-294002. However, LY-294002 did not inhibit serotonin- or PDBu-induced increases in myosin light chain phosphorylation or total O(2) consumption, suggesting that inhibition of contraction was not mediated by reversal or inhibition of the pathways that lead to smooth muscle activation and contraction. Treatment of vascular smooth muscle with LY-294002 increased the activity of cAMP-dependent protein kinase and increased the phosphorylation of the cAMP-dependent protein kinase substrate heat shock protein 20 (HSP20). These data suggest that activation of the PI3-kinase/Akt pathway in unstimulated smooth muscle may modulate vascular smooth muscle tone (allow agonist-induced contraction) through inhibition of the cyclic nucleotide/HSP20 pathway and suggest that cyclic nucleotide-dependent inhibition of contraction is dissociated from the myosin light chain contractile regulatory pathways.     

Heat-induced force suppression and HSP20 phosphorylation in swine carotid media.

In vascular smooth muscle, cyclic nucleotide-dependent phosphorylation of heat shock protein 20 (HSP20) on serine-16 (Ser16) has been suggested to cause force suppression, i.e., reduced force with only minimal myosin regulatory light chain (MRLC) dephosphorylation. We hypothesized that heat pretreatment also suppresses force by increasing HSP20 phosphorylation. After heat pretreatment of swine carotid artery at 44.5 degrees C for 4 h and reduction to 37 degrees C for 1 h, Ser16-HSP20 phosphorylation was increased and histamine-induced increases in contractile force were suppressed. Subsequent addition of nitroglycerin induced additive force suppression. Heat and nitroglycerin induced a similar relation between Ser16-HSP20 phosphorylation and force. Heat pretreatment induced a small, but significant, increase in total HSP20 immunostaining. These results demonstrate that vascular smooth muscle responds to thermal stress by increasing Ser16-HSP20 phosphorylation in addition to a possible small increase in total HSP20 concentration. The resulting heat-induced reduction in force should be considered "force suppression" because histamine-induced increases in MRLC phosphorylation were not significantly altered by heat pretreatment. These processes may bring about a resistance to contractile agonists, which could have clinical significance in conditions such as hyperthermia and/or sepsis with vasodilatory shock.     

Transduction of phosphorylated heat shock-related protein 20, HSP20, prevents vasospasm of human umbilical artery smooth muscle.

Activation of cyclic nucleotide-dependent signaling pathways inhibits agonist-induced contraction of most vascular smooth muscles except human umbilical artery smooth muscle (HUASM). This impaired vasorelaxation may contribute to complications associated with preeclampsia, intrauterine growth restriction, and preterm delivery. Cyclic nucleotide-dependent signaling pathways converge at the phosphorylation of the small heat shock-related protein HSP20, causing relaxation of vascular smooth muscle. We produced recombinant proteins containing a protein transduction domain linked to HSP20 (rTAT-HSP20). Pretreatment of HUASM with in vitro phosphorylated rTAT-HSP20 (rTAT-pHSP20) significantly inhibited serotonin-induced contraction, without a decrease in myosin light chain phosphorylation. rTAT-pHSP20 remained phosphorylated upon transduction into isolated HUASM as demonstrated by two-dimensional gel electrophoresis. Transduction of peptide analogs of phospho-HSP20 containing the phosphorylation site on HSP20 and phosphatase-resistant mimics of the phosphorylation site (S16E) also inhibited HUASM contraction. These data suggest that impaired relaxation of HUASM may result from decreased levels of phosphorylated HSP20. Protein transduction can be used to restore intracellular expression levels and the associated physiological response. Transduction of posttranslationally modified substrate proteins represents a proteomic-based therapeutic approach that may be particularly useful when the expression of downstream substrate proteins is downregulated.     

The small heat shock-related protein, HSP20, is a cAMP-dependent protein kinase substrate that is involved in airway smooth muscle relaxation

Activation of the cAMP/cAMP-dependent PKA pathway leads to relaxation of airway smooth muscle (ASM). The purpose of this study was to examine the role of the small heat shock-related protein HSP20 in mediating PKA-dependent ASM relaxation. Human ASM cells were engineered to constitutively express a green fluorescent protein-PKA inhibitory fusion protein (PKI-GFP) or GFP alone. Activation of the cAMP-dependent signaling pathways by isoproterenol (ISO) or forskolin led to increases in the phosphorylation of HSP20 in GFP but not PKI-GFP cells. Forskolin treatment in GFP but not PKI-GFP cells led to a loss of central actin stress fibers and decreases in the number of focal adhesion complexes. This loss of stress fibers was associated with dephosphorylation of the actin-depolymerizing protein cofilin in GFP but not PKI-GFP cells. To confirm that phosphorylated HSP20 plays a role in PKA-induced ASM relaxation, intact strips of bovine ASM were precontracted with serotonin followed by ISO. Activation of the PKA pathway led to relaxation of bovine ASM, which was associated with phosphorylation of HSP20 and dephosphorylation of cofilin. Finally, treatment with phosphopeptide mimetics of HSP20 possessing a protein transduction domain partially relaxed precontracted bovine ASM strips. In summary, ISO-induced phosphorylation of HSP20 or synthetic phosphopeptide analogs of HSP20 decreases phosphorylation of cofilin and disrupts actin in ASM, suggesting that one possible mechanism by which HSP20 mediates ASM relaxation is via regulation of actin filament dynamics.     

Selected contribution: HSP20 phosphorylation in nitroglycerin- and forskolin-induced sustained reductions in swine carotid media tone.

Cyclic nucleotide-induced relaxation of maximally activated arterial smooth muscle has two phases. 1) The initial relaxation transient is typically characterized by a rapid reduction in force associated with brief reductions in myoplasmic Ca(2+) concentration ([Ca(2+)](i)) and myosin regulatory light chain (MRLC) phosphorylation on serine (Ser)-19 (Ser(19)). 2) The sustained inhibitory response is typically associated with Ser(16) phosphorylation of heat shock protein 20 (HSP20) without sustained reductions in [Ca(2+)](i) or MRLC phosphorylation. We investigated whether the extent of Ser(16)-HSP20 phosphorylation quantitatively correlated with the sustained inhibitory response. With addition of nitroglycerin to histamine-stimulated swine carotid media, the initial relaxation transient was associated with a decrease in MRLC phosphorylation without an increase in Ser(16)-HSP20 phosphorylation. During the sustained phase of nitroglycerin-induced relaxation and during force redevelopment induced by washout of nitroglycerin in the continued presence of histamine, the level of Ser(16)-HSP20 phosphorylation, but not MRLC phosphorylation, correlated with inhibition of force. Forskolin, which increases cAMP concentration, also induced a sustained inhibitory response that was associated with increases in Ser(16)-HSP20 phosphorylation without reductions in MRLC phosphorylation levels. Forskolin increased Ser(16)-HSP20 phosphorylation to a greater extent and inhibited force more completely than that observed with nitroglycerin. Increases in Ser(16)-HSP20 phosphorylation correlated with the degree of force inhibition regardless of whether the relaxation was induced by nitroglycerin or forskolin. These data are consistent with the hypothesis that Ser(16)-HSP20 phosphorylation may be a cyclic nucleotide-dependent, yet MRLC phosphorylation-independent, inhibitor of smooth muscle contractile force.     

Phosphorylation by cyclic AMP-dependent protein kinase inhibits chaperone-like activity of human HSP22 <Emphasis Type="Italic">in vitro</Emphasis>

Human small heat shock protein with molecular mass 22 kD (HSP22, HspB8) contains two Ser residues (Ser24 and Ser57) in consensus sequence RXS and is effectively phosphorylated by cAMP-dependent protein kinase in vitro. Mutation S24D did not affect, whereas mutations S57D or S24,57D prevented phosphorylation of HSP22 by cAMP-dependent protein kinase thus indicating that Ser57 is the primary site of phosphorylation. Phosphorylation (or mutation) of Ser57 (or Ser24 and Ser57) resulted in changes of the local environment of tryptophan residues and increased HSP22 susceptibility to chymotrypsinolysis. Mutations mimicking phosphorylation decreased dissociation of HSP22 oligomer at low concentration without affecting its quaternary structure at high protein concentration. Mutations S24D, S57D, and especially S24,57D were accompanied by decrease of chaperone-like activity of HSP22 if insulin and rhodanase were used as substrates. Thus, phosphorylation by cAMP-dependent protein kinase affects the structure and decreases chaperone-like activity of HSP22 in vitro.     

Cyclosporine-induced renal artery smooth muscle contraction is associated with increases in the phosphorylation of specific contractile regulatory proteins

Cyclosporine A (CSA) is a type 2B phosphatase inhibitor which can induce contraction of renal artery smooth muscle. In this investigation, we examined the phosphorylation events associated with CSA-induced contraction of bovine renal artery smooth muscle. Contractile responses were determined in a muscle bath and the corresponding phosphorylation events were determined with whole cell phosphorylation and two-dimensional gel electrophoresis. CSA-induced contractions were associated with increases in the phosphorylation of the 20 kDa myosin light chains (MLC20) and different isoforms of the small heat shock protein, HSP27. Cyclic nucleotide-dependent relaxation of CSA-induced contractions was associated with increases in the phosphorylation of another small heat shock protein, HSP20, and decreases in the phosphorylation of the MLC20, and some isoforms of HSP27. These data suggest that CSA-induced contraction and relaxation of vascular smooth muscle is associated with increases in the phosphorylation of specific contractile regulatory proteins.     

Heat shock protein 20-mediated force suppression in forskolin-relaxed swine carotid artery

Increases in cyclic nucleotide levels induce smooth muscle relaxation by deactivation [reductions in myosin regulatory light chain (MRLC) phosphorylation (e.g., by reduced [Ca²⁺])] or force suppression (reduction in force without reduction in MRLC phosphorylation). Ser¹⁶-heat shock protein 20 (HSP20) phosphorylation is the proposed mediator of force suppression. We evaluated three potential hypotheses whereby Ser¹⁶-HSP20 phosphorylation could regulate smooth muscle force: 1) a threshold level of HSP20 phosphorylation could inactivate a thin filament as a whole, 2) phosphorylation of a single HSP20 could fully inactivate a small region of a thin filament, or 3) HSP20 phosphorylation could weakly inhibit myosin binding at either the thin- or thick-filament level. We tested these hypotheses by analyzing the dependence of force on Ser¹⁶-HSP20 phosphorylation in swine carotid media. First, we determined that swine HSP20 has a second phosphorylation site at Ser¹⁵⁷. Ser¹⁵⁷-HSP20 phosphorylation values were high and did not change during contractile activation or forskolin-induced relaxation. Forskolin significantly increased Ser¹⁶-HSP20 phosphorylation. The relationship between Ser¹⁶-HSP20 phosphorylation and force remained linear and was shifted downward in partially activated muscles relaxed with forskolin. Neither forskolin nor nitroglycerin induced actin depolymerization as detected using the F/G-actin ratio method in smooth muscle homogenates. These results suggest that force suppression does not occur in accordance with the first hypothesis (inactivation of a thin filament as a whole). Our data are more consistent with the second and third hypotheses that force suppression is mediated by full or partial inhibition of local myosin binding at the thin- or thick-filament level. cAMP; cGMP; nitric oxide; vascular smooth muscle     

The 204-kDa smooth muscle myosin heavy chain is phosphorylated in intact cells by casein kinase II on a serine near the carboxyl terminus

The heavy chain of smooth muscle myosin was found to be phosphorylated following immunoprecipitation from cultured bovine aortic smooth muscle cells. Of a variety of serine/threonine kinases assayed, only casein kinase II and calcium/calmodulin-dependent protein kinase II phosphorylated the smooth muscle myosin heavy chain to a significant extent in vitro. Two-dimensional maps of tryptic peptides derived from heavy chains phosphorylated in cultured cells revealed one major and one minor phosphopeptide. Identical tryptic peptide maps were obtained from heavy chains phosphorylated in vitro with casein kinase II but not with calcium/calmodulin-dependent protein kinase II. Of note, the 204-kDa smooth muscle myosin heavy chain but not the 200-kDa heavy chain isoform was phosphorylated by casein kinase II. Partial sequence of the tryptic phosphopeptides generated following phosphorylation by casein kinase II yielded Val-Ile-Glu-Asn-Ala-Asp-Gly-Ser*-Glu-Glu-Glu-Val. The Ser* represents the Ser(PO4) which is in an acidic environment, as is typical for casein kinase II phosphorylation sites. By comparison with the deduced amino acid sequence for rabbit uterine smooth muscle myosin (Nagai, R., Kuro-o, M., Babij, P., and Periasamy, M. (1989) J. Biol. Chem. 264, 9734-9737), we have localized the phosphorylated serine residue to the non-helical tail of the 204-kDa isoform of the smooth muscle myosin heavy chain. The ability of the 204-kDa isoform, but not the 200-kDa isoform, to serve as a substrate for casein kinase II suggests that these two isoforms can be regulated differentially.     

Effects of phosphorylation by protein kinase A on binding of catecholamines to the human tyrosine hydroxylase isoforms

Tyrosine hydroxylase (TyrH), the catalyst for the key regulatory step in catecholamine biosynthesis, is phosphorylated by cAMP-dependent protein kinase A (PKA) on a serine residue in a regulatory domain. In the case of the rat enzyme, phosphorylation of Ser40 by PKA is critical in regulating the enzyme activity; the effect of phosphorylation is to relieve the enzyme from inhibition by dopamine and dihydroxyphenylalanine (DOPA). There are four isoforms of human tyrosine hydroxylase (hTyrH), differing in the size of an insertion after Met30. The effects of phosphorylation by PKA on the binding of DOPA and dopamine have now been determined for all four human isoforms. There is an increase of about two-fold in the Kd value for DOPA for isoform 1 upon phosphorylation, from 4.4 to 7.4 microM; this effect decreases with the larger isoforms such that there is no effect of phosphorylation on the Kd value for isoform 4. Dopamine binds more much tightly, with Kd values less than 3 nM for all four unphosphorylated isoforms. Phosphorylation decreases the affinity for dopamine at least two orders of magnitude, resulting in Kd values of about 0.1 microM for the phosphorylated human enzymes, due primarily to increases in the rate constant for dissociation of dopamine. Dopamine binds about two-fold less tightly to the phosphorylated isoform 1 than to the other three isoforms. The results extend the regulatory model developed for the rat enzyme, in which the activity is regulated by the opposing effects of catecholamine binding and phosphorylation by PKA. The small effects on the relatively high Kd values for DOPA suggest that DOPA levels do not regulate the activity of hTyrH.     

Functional expression of NOS 1 in vascular smooth muscle

Substances that increase intracellular calcium concentration ([Ca(2+)](i)), such as serotonin, are known to induce vascular smooth muscle (VSM) contraction. However, increases in [Ca(2+)](i) also activate Ca(2+)/calmodulin-dependent nitric oxide synthases (NOS), which leads to increases in cGMP and activation of cGMP-dependent protein kinase (PKG). One recently identified substrate protein of PKG is the small heat shock protein, HSP20. The purpose of this study was to determine if serotonin activates a Ca(2+)-dependent NOS in VSM. Strips of bovine carotid arterial smooth muscle denuded of endothelium were stimulated with serotonin in the presence and absence of the nonspecific NOS inhibitor N-monomethyl-L-arginine (L-NMMA). Activation of NOS was determined by increases in cGMP and in the phosphorylation of HSP20. Immunohistochemical and Western blotting techniques were performed to identify specific NOS isoforms in bovine carotid arterial smooth muscle preparations. Serotonin stimulation led to significant increases in cGMP and in the phosphorylation of HSP20, which were inhibited by pretreatment with L-NMMA. Antibodies against NOS 1 stained the media of bovine carotid and human renal arteries, whereas antibodies against NOS 3 stained only the endothelium. Additionally, the conversion of radiolabeled L-arginine to L-citrulline NOS activity demonstrated a consistent amount of activity present in the endothelium-denuded smooth muscle preparations that was reduced by 99% with an NOS 1 specific inhibitor. Finally, an NOS 1 specific inhibitor, 7-nitroindazole, augmented contractions induced by high extracellular KCl. This study demonstrates that NOS 1 is present in VSM and may effect physiological contractile responses.     

Phosphorylation by protein kinase C of the 20,000-dalton light chain of myosin in intact and chemically skinned vascular smooth muscle

In the present study we tested the hypothesis that phosphorylation of the 20,000-dalton light chain subunit of smooth muscle myosin (LC20) by the calcium-activated and phospholipid-dependent protein kinase C regulates contraction of chemically-permeabilized (glycerinated) porcine carotid artery smooth muscle. Purified protein kinase C and oleic acid were used to phosphorylate LC20 in glycerinated muscles in the presence of a CaEGTA/EGTA buffer system (pCa 8) to prevent activation of myosin light chain kinase. Phosphorylation of the light chain to 1.3 mol of PO4/mol of LC20 did not stimulate contraction. Tryptic digests of glycerinated carotid artery LC20 contained two major phosphopeptides which contained phosphoserine but not phosphothreonine. Incubation of glycerinated muscles with calcium (20 microM) and calmodulin (10 microM) resulted in contraction and LC20 phosphorylation to 1.1 mol of PO4/mol of LC20; tryptic digests of LC20 from these muscles contained a single phosphopeptide which could be distinguished by phosphopeptide mapping from the two phosphopeptides derived from muscles phosphorylated with protein kinase C. Further phosphorylation of Ca2+/calmodulin-activated muscles to 2.0 mol of PO4/mol of LC20, by incubation with protein kinase C, had no effect on either the level of isometric force or the lightly-loaded shortening velocity (after-load = 0.1 peak active force); removal of Ca2+ and calmodulin, but not protein kinase C and oleic acid, resulted in normal relaxation in spite of maintained phosphorylation to 1.2 mol of PO4/mol of LC20. Comparison of LC20 phosphopeptide maps from glycerinated muscles incubated with protein kinase C plus Ca2+/calmodulin (2.0 mol of PO4/mol of LC20) to maps from intact muscles stimulated with 10(-6) M phorbol 12,13-dibutyrate (0.05 mol of PO4/mol of LC20) showed that the same three phosphopeptides were present in both the intact and glycerinated muscles. These findings show that phosphorylation of LC20 by protein kinase C in glycerinated muscles to levels at least 40 times higher than those present during contraction of intact, phorbol ester-stimulated muscles does not activate contraction nor does it significantly modify the contraction of smooth muscle which occurs in response to the Ca2+/calmodulin-dependent phosphorylation of Ser19 by myosin light chain kinase.     

Interaction of phosphorylated tyrosine hydroxylase with 14-3-3 proteins: evidence for a phosphoserine 40-dependent association

Tyrosine hydroxylase (TH) has been reported to require binding of 14-3-3 proteins for optimal activation by phosphorylation. We examined the effects of phosphorylation at Ser19, Ser31 and Ser40 of bovine TH and human TH isoforms on their binding to the 14-3-3 proteins BMH1/BMH2, as well as 14-3-3 zeta and a mixture of sheep brain 14-3-3 proteins. Phosphorylation of Ser31 did not result in 14-3-3 binding, however, phosphorylation of TH on Ser40 increased its affinity towards the yeast 14-3-3 isoforms BMH1/BMH2 and sheep brain 14-3-3, but not for 14-3-3 zeta. On phosphorylation of both Ser19 and Ser40, binding to the 14-3-3 zeta isoform also occurred, and the binding affinity to BMH1 and sheep brain 14-3-3 increased. Both phosphoserine-specific antibodies directed against the 10 amino acids surrounding Ser19 or Ser40 of TH, and the phosphorylated peptides themselves, inhibited the association between phosphorylated TH and 14-3-3 proteins. This was also found when heparin was added, or after proteolytic removal of the N-terminal 37 amino acids of Ser40-phosphorylated TH. Binding of BMH1 to phosphorylated TH decreased the rate of dephosphorylation by protein phosphatase 2A, but no significant change in enzymatic activity was observed in the presence of BMH1. These findings further support a role for 14-3-3 proteins in the regulation of catecholamine biosynthesis and demonstrate isoform specificity for both TH and 14-3-3 proteins.     

Phosphorylation of regulatory light chain a (RLC-a) in smooth muscle myosin of scallop, Patinopecten yessoensis

One of the two regulatory light chains, RLC-a, of scallop smooth muscle myosin was fully phosphorylated by myosin light chain kinase of chicken gizzard muscle. The residue phosphorylated was Ser. It may be the Ser at number 11 from the N-terminal. The sequence of 9 residues around the Ser-11, QRATSNVFA, is identical with that around the phosphorylatable Ser of LC20 of chicken gizzard myosin. RLC-a was also phosphorylated slowly by cAMP-dependent protein kinase. The phosphorylation of RLC-a may be involved in the regulatory system for the catch contraction of scallop muscle.     

Longer muscle lengths recapitulate force suppression in swine carotid artery

Cyclic nucleotide can relax arterial smooth muscle without reductions in myosin regulatory light chain (MRLC) phosphorylation, a process termed force suppression. Smooth muscle contractile force also depends on tissue length. It is not known how tissue length affects force suppression. Swine carotid artery rings were equilibrated at various lengths (as a fraction of L(o), the optimal length for force development). They were then frozen during contractile activation with or without forskolin-induced relaxation. Frozen tissue homogenates were then analyzed for Ser(19)-MRLC phosphorylation and Ser(16)-heat shock protein 20 (HSP20) phosphorylation (HSP20 is the proposed mediator of force suppression). Higher values of MRLC phosphorylation were required to induce a histamine contraction at longer tissue lengths. At 1.4 L(o), the dependence of force on MRLC phosphorylation observed with histamine stimulation alone was shifted to the right, a response similar to that observed during force suppression at 1.0 L(o). The rightward shift in the dependence of force on MRLC phosphorylation seen with histamine stimulation alone at 1.4 L(o) was not associated with increased HSP20 phosphorylation. Addition of forskolin to histamine-stimulated tissues at 1.4 L(o) induced a relaxation associated with increased HSP20 phosphorylation and reduced MRLC phosphorylation, i.e., there was no additional force suppression. At shorter tissue lengths (0.6 L(o)), the dependence of force on MRLC phosphorylation with histamine stimulation alone was steep, a response similar to that observed during normal contractile activation at 1.0 L(o). Addition of forskolin induced force suppression at 0.6 L(o). The sensitivity of swine carotid to the concentration of histamine was greater at longer tissue lengths compared with shorter tissue lengths, suggesting a physiological mechanism to restore optimal tissue length. These data suggest that longer tissue lengths induced a force suppression-like state that was 1) not additive with forskolin and 2) not associated with HSP20 phosphorylation. Further research is required to determine this length-dependent mechanism.     

Mammal-specific, ERK-dependent, caldesmon phosphorylation in smooth muscle. Quantitation using novel anti-phosphopeptide antibodies.

Extracellular signal-regulated kinases (ERKs) phosphorylate the high molecular mass isoform of the actin-binding protein caldesmon (h-CaD) at two sites (Ser(759) and Ser(789)) during smooth muscle stimulation. To investigate the role of phosphorylation at these sites, antibodies were generated against phosphopeptides analogous to the sequences around Ser(759) and Ser(789). Affinity-purified antibodies were phosho- and sequence-specific. The major site of phosphorylation in h-CaD in porcine carotid arterial muscle strips was at Ser(789); however, the amount of phosphate did not vary appreciably with either KCl or phorbol ester stimulation. Phosphorylation at Ser(759) of h-CaD was almost undetectable (<0.005 mol of phosphate/mol of protein). Moreover, phosphorylation of the low molecular mass isoform of the protein (l-CaD) at the site analogous to Ser(789) was greater in serum-stimulated cultured smooth muscle cells than in serum-starved cells. Serum-stimulated l-CaD phosphorylation was attenuated by the protein kinase inhibitor PD98059. These data 1) identify Ser(789) of h-CaD as the major site of ERK-dependent phosphorylation in carotid arteries; 2) show that the level of phosphorylation at Ser(789) is relatively constant following carotid arterial muscle stimulation, despite an increase in total protein phosphate content; and 3) suggest a functional role for ERK-dependent l-CaD phosphorylation in cell division.     

Heterogeneity in smooth muscle cell population accumulating in the neointimas and the media of poststenotic dilatation of the rabbit carotid artery.

Rabbit smooth muscles contain at least three types of myosin heavy chain (MHC) isoforms; SM1, SM2 and SMemb (NMHC-B), the expression of which is developmentally regulated. We have recently reported that smooth muscles with the embryonic phenotype accumulate in the neointimas produced by endothelial denudation or high-cholesterol feeding. In this study, we examined MHC isoform expression in the neointimas and the media of poststenotic dilatation of the rabbit carotid artery, and determined the phenotype of the smooth muscle cell in the dilated segment. We report here that neointimal cells in the dilated segment are smooth muscle cells with the embryonic phenotype as previously reported in our ballooning-injury study. The medial smooth muscles, however, are composed of heterogeneous population of smooth muscles which differ in stage of differentiation as determined by the MHC isoform expression. These results indicate that MHC isoforms are useful molecular markers to identify abnormally proliferating smooth muscles in diseased arteries and to understand the process of atherogenesis occurring following vascular injury.