The apoptotic regulatory protein ARC (apoptosis repressor with caspase recruitment domain) prevents oxidant stress-mediated cell death by preserving mitochondrial function.

ARC is an apoptotic regulatory protein expressed almost exclusively in myogenic cells. It contains a caspase recruitment domain (CARD) through which it has been shown to block the activation of some initiator caspases. Because ARC also blocks caspase-independent events associated with apoptosis, such as hypoxia-induced cytochrome c release, we examined its role in cell death triggered by exposure to hydrogen peroxide (H(2)O(2)) in the myogenic cell line, H9c2. Cell death in this model was caspase-independent and characterized by dose-dependent reduction in ARC expression accompanied by disruption of the mitochondrial membrane potential (Delta psi(m)) and loss of plasma membrane integrity, typical of necrotic cell death. Ectopic expression of ARC prevented both H(2)O(2)-induced mitochondrial dysfunction and cell death without affecting the stress kinase response, suggesting that ARCs protective effects were downstream of early signaling events and not due to quenching of H(2)O(2). ARC was also effective in blocking H(2)O(2)-induced loss of membrane integrity and/or disruption of Delta psi(m) in two human cell lines in which it is not normally expressed. These results demonstrate that, in addition to its ability to block caspase-dependent and -independent events in apoptosis, ARC also prevents necrosis-like cell death via the preservation of mitochondrial function.

Requirement for p38 and p44/p42 mitogen-activated protein kinases in RAGE-mediated nuclear factor-kappaB transcriptional activation and cytokine secretion.

Advanced glycation end product (AGE) activation of the signal-transducing receptor for AGE (RAGE) has been linked to a proinflammatory phenotypic change within cells. However, the precise intracellular signaling pathways involved have not been elucidated. We demonstrate here that human serum albumin modified with N(epsilon)-(carboxymethyl)lysine (CML), a major AGE adduct that progressively accumulates with aging, diabetes, and renal failure, induced nuclear factor (NF)-kappaB-driven reporter gene expression in human monocytic THP-1 cells. The NF-kappaB response was blocked with a synthetic peptide corresponding to the putative ligand-binding domain of RAGE, with anti-RAGE antiserum, and by coexpression of truncated receptors lacking the intracellular domain. Signal transduction from RAGE to NF-kappaB involved the generation of reactive oxygen species, since reporter gene expression was blocked with the antioxidant N-acetyl-L-cysteine. CML-modified albumin produced rapid transient activation of tyrosine phosphorylation, extracellular signal-regulated kinase 1 and 2, and p38 mitogen-activated protein kinase (MAPK), but not c-Jun NH(2)-terminal kinase. RAGE-mediated NF-kappaB activation was suppressed by the selective p38 MAPK inhibitor SB203580 and by coexpression of a kinase-dead p38 dominant-negative mutant. Activation of NF-kappaB by CML-modified albumin increased secretion of proinflammatory cytokines (tumor necrosis factor-alpha, interleukin-1beta, and monocyte chemoattractant protein-1) severalfold, and inhibition of p38 MAPK blocked these increases. These results indicate that p38 MAPK activation mediates RAGE-induced NF-kappaB-dependent secretion of proinflammatory cytokines and suggest that accelerated inflammation may be a consequence of cellular activation induced by this receptor.

Differential effect of MLC kinase in TNF-alpha-induced endothelial cell apoptosis and barrier dysfunction.

Tumor necrosis factor (TNF)-alpha is released in acute inflammatory lung syndromes linked to the extensive vascular dysfunction associated with increased permeability and endothelial cell apoptosis. TNF-alpha induced significant decreases in transcellular electrical resistance across pulmonary endothelial cell monolayers, reflecting vascular barrier dysfunction (beginning at 4 h and persisting for 48 h). TNF-alpha also triggered endothelial cell apoptosis beginning at 4 h, which was attenuated by the caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone. Exploring the involvement of the actomyosin cytoskeleton in these important endothelial cell responses, we determined that TNF-alpha significantly increased myosin light chain (MLC) phosphorylation, with prominent stress fiber and paracellular gap formation, which paralleled the onset of decreases in transcellular electrical resistance and enhanced apoptosis. Reductions in MLC phosphorylation by the inhibition of either MLC kinase (ML-7, cholera toxin) or Rho kinase (Y-27632) dramatically attenuated TNF-alpha-induced stress fiber formation, indexes of apoptosis, and caspase-8 activity but not TNF-alpha-induced barrier dysfunction. These studies indicate a central role for the endothelial cell cytoskeleton in TNF-alpha-mediated apoptosis, whereas TNF-alpha-induced vascular permeability appears to evolve independently of contractile tension generation.

Apoptosis in cardiac disease--what is it--how does it occur.

This review will present a summary of a description of apoptotic pathways in the heart, followed by ways to measure it and the experimental and clinical evidence for the role of apoptosis in cardiac disease. An evaluation of the effectiveness of pharmacological and other therapeutic interventions in the prevention of apoptosis in the context of cardiac disease will also be presented.

Hypoxia-induced Haem Oxygenase-1 gene expression in neonatal rat cardiac myocytes.

BACKGROUND: Haem oxygenase (HO-1), a heat shock or stress protein, is a rate-limiting enzyme in the conversion of pro-oxidant haem to biliverdin and carbon monoxide (CO). The products of haem catabolism serve regulatory and protective functions. Previous studies have shown that hypoxia induces HO-1 expression in cardiac myocytes. Accordingly, we investigated whether hypoxia-induced HO-1 expression is accompanied by increased CO production in cultured neonatal rat cardiac myocytes, and whether protein kinase C (PKC) is involved in hypoxia induced HO-1 gene expression. METHODS AND RESULTS: Expression of HO-1 in hypoxia-treated cells was examined by using northern and western blotting, and immunofluorescent staining. The level of HO-1 mRNA at 24 and 48 h was increased after the onset of hypoxia, with corresponding increase in the HO-1 protein level (6.7- and 8.7-fold at 24 and 48 h of hypoxia, respectively). HO-1 protein was colocalised with sarcomeric alpha-actin in hypoxic myocytes. Hypoxia also significantly increased the production of CO by 2.5- and 8-fold at 24 and 48 h, respectively. Under normoxic conditions, activation of PKC by phorbol-12-myristate-13-acetate (PMA; 100 nmol/L) markedly increased HO-1 gene expression, while inhibition of PKC activity by calphostin C (100 nmol/L) blocked hypoxia-induced HO-1 gene expression in cardiac myocytes. CONCLUSIONS: These results demonstrate that hypoxia markedly induces HO-1 expression and increases the production of CO in cardiac myocytes. This hypoxic response is attributed, at least in part, to activation of PKC. Increased HO-1 expression and resultant CO production may be beneficial with respect to protection of cardiac myocytes under hypoxic conditions.

beta-blockade prevents sustained metalloproteinase activation and diastolic stiffening induced by angiotensin II combined with evolving cardiac dysfunction.

Angiotensin II (Ang II)-mediated sympathostimulation may worsen the progression of cardiac failure, although the nature and mechanisms of such interactions are largely unknown. We previously demonstrated that Ang II combined with evolving cardiodepression (48-hour tachycardia pacing, 48hP) induces marked chamber stiffening and increases metalloproteinases (MMPs). Here, we test the hypothesis that both abnormalities stem from sympathostimulatory effects of Ang II. Forty-eight dogs were instrumented to serially assess conscious ventricular mechanics, MMP abundance and activity, and myocardial histopathology. 48hP combined with 5 days of Ang II (15+/-5 ng. kg(-1). min(-1) IV) more than doubled chamber stiffness (end-diastolic pressure >25 mm Hg, P<0.001), whereas stiffness was unchanged by Ang II or 48hP alone. In vitro and in situ zymography revealed increased MMP abundance and activity (principally 92-kDa gelatinase) from Ang II+48hP. Both stiffening and MMP changes were prevented by cotreatment with high-dose atenolol (which nearly fully inhibited isoproterenol-induced inotropy) but not partial beta-blockade. Myocellular damage with fibroblast/neutrophil infiltration from Ang II+48hP was also inhibited by high- but not low-dose atenolol, whereas collagen content was not elevated with either dose. These data support a role of sympathostimulation by Ang II in modulating myocardial MMP abundance and activity and diastolic stiffening in evolving heart failure and suggest a novel mechanism by which beta-blockade may limit chamber remodeling and diastolic dysfunction.

The beta(2)-adrenergic receptor delivers an antiapoptotic signal to cardiac myocytes through G(i)-dependent coupling to phosphatidylinositol 3'-kinase.

Recent studies have shown that chronic beta-adrenergic receptor (beta-AR) stimulation alters cardiac myocyte survival in a receptor subtype-specific manner. We examined the effect of selective beta(1)- and beta(2)-AR subtype stimulation on apoptosis induced by hypoxia or H(2)O(2) in rat neonatal cardiac myocytes. Although neither beta(1)- nor beta(2)-AR stimulation had any significant effect on the basal level of apoptosis, selective beta(2)-AR stimulation protected myocytes from apoptosis. beta(2)-AR stimulation markedly increased mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MAPK/ERK) activation as well as phosphatidylinositol-3'-kinase (PI-3K) activity and Akt/protein kinase B phosphorylation. beta(1)-AR stimulation also markedly increased MAPK/ERK activation but only minimally activated PI-3K and Akt. Pretreatment with pertussis toxin blocked beta(2)-AR-mediated protection from apoptosis as well as the beta(2)-AR-stimulated changes in MAPK/ERK, PI-3K, and Akt/protein kinase B. The selective PI-3K inhibitor, LY 294002, also blocked beta(2)-AR-mediated protection, whereas inhibition of MAPK/ERK activation at an inhibitor concentration that blocked agonist-induced activation but not the basal level of activation had no effect on beta(2)-AR-mediated protection. These findings demonstrate that beta(2)-ARs activate a PI-3K-dependent, pertussis toxin-sensitive signaling pathway in cardiac myocytes that is required for protection from apoptosis-inducing stimuli often associated with ischemic stress.

ARC inhibits cytochrome c release from mitochondria and protects against hypoxia-induced apoptosis in heart-derived H9c2 cells.

Ischemia induces apoptosis as well as necrosis of cardiac myocytes. We recently reported the cloning of a cDNA that encodes an apoptotic inhibitor, ARC, that is expressed predominantly in cardiac and skeletal muscle. In the present study, we examined the ability of ARC to protect rat embryonic heart-derived H9c2 cells from apoptosis induced by hypoxia, a component of ischemia. We found that H9c2 cells express ARC and that exposure to hypoxia substantially reduces ARC expression while inducing apoptosis. Transfected H9c2 cells in which cytosolic ARC protein levels remain elevated during hypoxia were significantly more resistant to hypoxia-induced apoptosis than parental H9c2 cells or H9c2 cells transfected with a control vector. Loss of endogenous ARC in the cytosol of H9c2 cells was associated with translocation of ARC from the cytosol to intracellular membranes, release of cytochrome c from the mitochondria, activation of caspase-3, poly(ADP-ribose)polymerase (PARP) cleavage, and DNA fragmentation. All of these events were inhibited in H9c2 cells overexpressing ARC when compared with control cells. In contrast, caspase inhibitors prevented PARP cleavage but not cytochrome c release, suggesting that exogenously expressed ARC acts upstream of caspase activation in this model of apoptosis. These results demonstrate that ARC can protect heart myogenic H9c2 cells from hypoxia-induced apoptosis, and that ARC prevents cytochrome c release by acting upstream of caspase activation, perhaps at the mitochondrial level.

A molecular mechanism of integrin crosstalk: alphavbeta3 suppression of calcium/calmodulin-dependent protein kinase II regulates alpha5beta1 function.

Many cells express more than one integrin receptor for extracellular matrix, and in vivo these receptors may be simultaneously engaged. Ligation of one integrin may influence the behavior of others on the cell, a phenomenon we have called integrin crosstalk. Ligation of the integrin alphavbeta3 inhibits both phagocytosis and migration mediated by alpha5beta1 on the same cell, and the beta3 cytoplasmic tail is necessary and sufficient for this regulation of alpha5beta1. Ligation of alpha5beta1 activates the calcium- and calmodulin-dependent protein kinase II (CamKII). This activation is required for alpha5beta1-mediated phagocytosis and migration. Simultaneous ligation of alphavbeta3 or expression of a chimeric molecule with a free beta3 cytoplasmic tail prevents alpha5beta1-mediated activation of CamKII. Expression of a constitutively active CamKII restores alpha5beta1 functions blocked by alphavbeta3-initiated integrin crosstalk. Thus, alphavbeta3 inhibition of alpha5beta1 activation of CamKII is required for its role in integrin crosstalk. Structure-function analysis of the beta3 cytoplasmic tail demonstrates a requirement for Ser752 in beta3-mediated suppression of CamKII activation, while crosstalk is independent of Tyr747 and Tyr759, implicating Ser752, but not beta3 tyrosine phosphorylation in initiation of the alphavbeta3 signal for integrin crosstalk.

Myocardial retinoid X receptor, thyroid hormone receptor, and myosin heavy chain gene expression in the rat during adult aging.

Although previous studies have shown that cardiac myosin heavy chain (MHC) composition undergoes a switch from the alpha- to beta-isoform in the heart during adult aging, the underlying mechanisms responsible for this switch are unknown. Cardiac MHC gene expression is regulated, in part, by thyroid hormone responsive elements present in the regulatory control regions of the alpha- and beta-MHC genes. Age-associated changes in the expression of thyroid hormone receptors (THRs) and/or retinoid X receptors (RXRs), the heterodimeric partner for THRs, could explain the age-associated changes in MHC expression. Accordingly, we measured mRNA levels for the cardiac muscle MHCs and the rat THR and RXR genes in the left ventricles of Wistar rats at 2, 6, and 24 months of age. Although there were no significant changes in RXR alpha or RXR beta mRNA levels with age, both alpha 1 and alpha 2 THR mRNA levels decreased significantly between 2 and 6 months of age. During this same time period, the mRNA levels for alpha-MHC declined by more than half, whereas beta-MHC mRNA levels remained low and unchanged. On the other hand, between 6 and 24 months, when mRNA levels for beta-MHC increased and alpha-MHC continued to decrease, there was a significant decline in THR beta 1 and RXR gamma mRNA levels accompanied by a reduction in the THR beta 1 and RXR gamma protein levels. These data show a pattern of change that suggests that the decline in alpha-MHC gene expression may be biphasic and due to a decline in alpha 1 (and possibly alpha 2) THR levels between 2 and 6 months of age and a decline in THR beta 1 and RXR gamma levels at later stages. In contrast, the increase in beta-MHC gene expression was associated only with the changes in THR beta 1 and RXR gamma mRNA and protein levels.

Age-related changes in the signaling and function of vascular smooth muscle cells.

Aging is an independent risk factor for the development of atherosclerosis, a vascular abnormality that plays a significant role in the development of many cardiovascular disorders. Animal experiments have demonstrated that aging predisposes the vasculature to advanced atherosclerotic disease and vessel injury and that this predisposition is a function of age-associated changes in the vessel wall itself. Because vascular smooth muscle cells play important roles in the pathogenesis of many vascular disorders, identifying age-associated differences in the way these cells respond to extracellular clues has been an area of active research. Currently, the most remarkable differences in intracellular signaling between vascular smooth muscle cells isolated from young and old animals are related to the control of cell migration through the CamKII pathways and the accelerated transition of older vascular smooth muscle cells from the contractile to the synthetic phenotype. These differences may be due to alternative signaling pathways revealed by the inability of older cells to respond to inhibitors, such as transforming growth factor (TGF)-beta1, or to altered interactions with the extracellular matrix resulting from age-associated shifts in integrin expression or changes in the matrix composition of blood vessels. The exact role that these alterations have in explaining age-associated differences in the response of the vessel wall to injury and its increased susceptibility to developing advanced atherosclerotic lesions remains to be determined but will be guided by studies on intracellular signaling mechanisms.

Adenovirus-mediated gene transfer of the human tissue inhibitor of metalloproteinase-2 blocks vascular smooth muscle cell invasiveness in vitro and modulates neointimal development in vivo.

BACKGROUND: Endovascular injury induced by balloon withdrawal leads to the increased activation of matrix metalloproteinases (MMPs) in the vascular wall, allowing smooth muscle cells (SMCs) to digest the surrounding extracellular matrix (ECM) and migrate from the media into the intima. The objective of this study was to examine the effects of a replication-deficient adenovirus carrying the cDNA for human tissue inhibitor of metalloproteinase-2 (AdCMV.hTIMP-2) on SMC function in vitro and neointimal development in the injured rat carotid artery. METHODS AND RESULTS: Infection of cultured rat aortic SMCs at a multiplicity of infection of 100 with AdCMV.hTIMP-2 resulted in high-level expression of hTIMP-2 mRNA and protein secretion into the medium. Conditioned media (CM) from AdCMV. hTIMP-2-infected but not control virus (AdCMV.null or AdCMV. betagal)-infected SMCs inhibited MMP-2 activity on gelatin zymograms as well as the chemoattractant-directed migration of SMCs across reconstituted basement membrane proteins in the Boyden chamber assay. In contrast, AdCMV.hTIMP-2 CM had no effect on chemoattractant-directed migration of SMCs occurring in the absence of an ECM barrier or on the proliferation of cultured neointimal SMCs. Delivery of AdCMV.hTIMP-2 (2.5x10(9) pfu) to the carotid artery wall at the time of balloon withdrawal injury inhibited SMC migration into the intima by 36% (P<0.05) at 4 days and neointimal area by 53% (P<0.01) at 8 days and by 12% (P=NS) at 21 days after injury. AdCMV.hTIMP-2 had no effect on medial area. CONCLUSIONS: Adenovirus-mediated hTIMP-2 gene transfer inhibits SMC invasiveness in vitro and in vivo and delays neointimal development after carotid injury.

Enhanced expression of p53 and apoptosis induced by blockade of the vacuolar proton ATPase in cardiomyocytes.

Activation of the vacuolar proton ATPase (VPATPase) has been implicated in the prevention of apoptosis in neutrophils and adult cardiac myocytes. To determine the role of the VPATPase in apoptosis of cardiac myocytes, we used a potent and specific inhibitor of the VPATPase, bafilomycin A1. Bafilomycin A1 alone caused increased DNA laddering of genomic DNA and increased nuclear staining for fragmented DNA in neonatal cardiomyocyte apoptosis in a dose- and time-dependent manner. Intracellular acidification in cardiac myocytes was also observed after 18 h of bafilomycin A1 treatment. Accordingly, bafilomycin A1-treated myocytes also showed increased accumulation of p53 protein and p53-dependent transactivation of gene expression, including a persistent upregulation of p21/wild-type p53 activated fragment 1/cyclin kinase inhibitor protein-1 mRNA. The bafilomycin A1-induced increase in p53 protein levels was accompanied by a marked increase in p53 mRNA accumulation. In contrast, cardiac fibroblasts treated with bafilomycin A1 showed no change in p53 protein expression or pHi and did not undergo apoptosis even after 24 h of treatment. Our data suggest that blockade of the VPATPase induces apoptotic cell death of cardiac myocytes and that this may occur through a p53-mediated apoptotic pathway.

Synergistic exacerbation of diastolic stiffness from short-term tachycardia-induced cardiodepression and angiotensin II.

Synergistic interaction between angiotensin II (Ang II) and evolving cardiodepression may play an important role in worsening chamber function, particularly in diastole. To test this hypothesis, Ang II was infused at 10 or 17 ng.kg(-1).min(-1) in 18 conscious dogs 4 days before and during induction of subacute cardiodepression by 48-hour tachypacing. The lower dose yielded negligible systemic pressure changes. Twelve additional animals served as paced-only controls. Pressure-dimension relations were recorded, and serial endocardial biopsies were obtained to assess histological and metalloproteinase (MMP) changes. Forty-eight-hour pacing alone depressed systolic function but had little effect on diastolic stiffness. Ang II alone only modestly raised diastolic stiffness at both doses and enhanced contractility at the higher dose. These changes recovered toward baseline after a 7-day infusion. However, Ang II (at either dose) combined with 48-hour pacing markedly increased ventricular stiffness (110+/-26% over baseline) and end-diastolic pressure (22+/-1.7 mm Hg). In contrast, pacing-induced inotropic and relaxation abnormalities were not exacerbated by Ang II. Zymography revealed MMP activation (72- and 92-kD gelatinases and 52-kDa caseinase) after a 4-day Ang II infusion (at both doses), which persisted during pacing. Tachypacing initiated 24 hours after cessation of a 7-day Ang II infusion also resulted in diastolic stiffening and corresponded with MMP reactivation. Ang II also induced myocyte necrosis, inflammation, and subsequent interstitial fibrosis, but these changes correlated less with chamber mechanics. Thus, Ang II amplifies and accelerates diastolic dysfunction when combined with evolving cardiodepression. This phenomenon may also underlie Ang II influences in late-stage cardiomyopathy, when chamber distensibility declines.

Increased expression of membrane-type matrix metalloproteinase and preferential localization of matrix metalloproteinase-2 to the neointima of balloon-injured rat carotid arteries.

BACKGROUND: Remodeling of the injured vascular wall is dependent on the action of several extracellular proteases. Previous studies have shown that expression of matrix metalloproteinases (MMP-2 and MMP-9) is upregulated after vascular injury and that MMP-2 is required for the migration of cultured vascular smooth muscle cells across complex extracellular matrix barriers. The present study examined changes in the expression of membrane-type metalloproteinase (MT-MMP-1), a putative regulator of MMP-2, in the tissue localization of MMP-2, and in the expression of activated and latent forms of MMP-2 and the tissue inhibitor of metalloproteinases, TIMP-2, in rat carotid arteries subjected to balloon catheter injury. METHODS AND RESULTS: MT-MMP-1 mRNA levels increased sixfold after 3 days of injury, coinciding with an increase in MMP-2 activation assessed by gelatin zymography. Western blotting and gelatin zymography showed an increase in MMP-2 protein levels beginning 5 to 7 days after injury; immunocytochemistry and Western blotting showed that the increase occurred preferentially in the developing neointima. CONCLUSIONS: These results show that increased expression of MT-MMP-1 and activation of MMP-2 occurs early after injury to the rat carotid artery and that at later times MMP-2 is preferentially localized to the developing neointima.

Regulation of vascular smooth muscle migration by mitogen-activated protein kinase and calcium/calmodulin-dependent protein kinase II signaling pathways.

Platelet-derived growth factor BB (PDGF BB) activation of the mitogen-activated protein kinases (MAPK), ERK1 and ERK2, has been shown to be necessary for mitogen-stimulated proliferation, but its role in regulating cell migration and its relationship to other chemotactic signaling events, such as CamKII activation, has not been defined. Using a modified Boyden chamber apparatus, we tested the effects of a selective inhibitor of the upstream activator of ERK1/2, MEK1, on PDGF-stimulated rat aortic vascular smooth muscle cells (VSMCs) alone and in combination with KN62, a selective inhibitor of CamKII. The MEK1 inhibitor, PD98059, caused a dose-dependent reduction in ERK2 activity that paralleled a decrease in migration up to 60%. This inhibition of migration was similar to that seen with KN62 and the combined effects of both inhibitors were non-additive. Although KN62 did not affect ERK2 activity in response to PDGF, PD98059 markedly inhibited PDGF-stimulated CamKII activity, suggesting that activation of CamKII by PDGF was dependent on ERK activity and that the effects of ERK inhibition on migration may be mediated through its ability to inhibit CamKII activity. To directly test this, VSMCs were infected with a recombinant adenovirus expressing constitutively activated CamKII. Infection reversed the inhibitory effects of KN62 on migration, but had no effect on the inhibition of migration seen with PD98059. These results suggest that while MAPK may act upstream of CamKII to control its activation in response to PDGF, it also regulates migration independently of CamKII activation.

Identification of specific mRNAs affected by treatments producing long-term facilitation in Aplysia.

Neural correlates of long-term sensitization of defensive withdrawal reflexes in Aplysia occur in sensory neurons in the pleural ganglia and can be mimicked by exposure of these neurons to serotonin (5-HT). Studies using inhibitors indicate that transcription is necessary for production of long-term facilitation by 5-HT. Several mRNAs that change in response to 5-HT have been identified, but the molecular events responsible for long-term facilitation have not yet been fully described. To detect additional changes in mRNAs, we investigated the effects of 5-HT (1.5 hr) on levels of mRNA in pleural-pedal ganglia using in vitro translation. Four mRNAs were affected by 5-HT, three of which were identified as calmodulin (CaM), phosphoglycerate kinase (PGK), and a novel gene product (protein 3). Using RNase protection assays, we found that 5-HT increased all three mRNAs in the pleural sensory neurons. CaM and protein 3 mRNAs were also increased in the sensory neurons by sensitization training. Furthermore, stimulation of peripheral nerves of pleural-pedal ganglia, an in vitro analog of sensitization training, increased the incorporation of labeled amino acids into CaM, PGK, and protein 3. These results indicate that increases in CaM, PGK, and protein 3 are part of the early response of sensory neurons to stimuli that produce long-term facilitation, and that CaM and protein 3 could have a role in the generation of long-term sensitization.

Captopril modifies gene expression in hypertrophied and failing hearts of aged spontaneously hypertensive rats.

The spontaneously hypertensive rat (SHR) exhibits a transition from stable compensated left ventricular (LV) hypertrophy to heart failure (HF) at a mean age of 21 months that is characterized by a decrease in alpha-myosin heavy chain (alpha-MHC) gene expression and increases in the expression of the atrial natriuretic factor (ANF), pro-alpha1(III) collagen, and transforming growth factor beta1 (TGF-beta1) genes. We tested the hypotheses that angiotensin-converting enzyme inhibition (ACEI) in SHR would prevent and reverse HF-associated changes in gene expression when administered prior to and after the onset of HF, respectively. We also investigated the effect of ACEI on circulating and cardiac components of the renin-angiotensin system. ACEI (captopril 2 g/L in the drinking water) was initiated at 12, 18, and 21 months of age in SHR without HF and in SHR with HF. Results were compared with those of age-matched normotensive Wistar-Kyoto (WKY) rats, and to untreated SHR with and without evidence of HF. ACEI initiated prior to failure prevented the changes in alpha-MHC, ANF, pro-alpha1(III) collagen, and TGF-beta1 gene expression that are associated with the transition to HF. ACEI initiated after the onset of HF lowered levels of TGF-beta1 mRNA by 50% (P<.05) and elevated levels of alpha-MHC mRNA two- to threefold (P<.05). Circulating levels of renin and angiotensin I were elevated four- to sixfold by ACEI, but surprisingly, plasma levels of angiotensin II were not reduced. ACEI increased LV renin mRNA levels in WKY and SHR by two- to threefold but did not influence LV levels of angiotensinogen mRNA. The results suggest that the anti-HF benefits of ACEI in SHR may be mediated, at least in part, by effects on the expression of specific genes, including those encoding alpha-MHC, ANF, TGF-beta1, pro-alpha1(III) collagen, and renin-angiotensin system components.

Rapamycin inhibits alpha 1-adrenergic receptor-stimulated cardiac myocyte hypertrophy but not activation of hypertrophy-associated genes. Evidence for involvement of p70 S6 kinase.

The 70-kD S6 kinase (p70S6K) has been implicated in the regulation of protein synthesis in many cell types and in the angiotensin II-stimulated hypertrophy of cardiac myocytes. Our purpose was to determine whether p70S6K plays a role in cardiomyocyte hypertrophy induced by the alpha 1-adrenergic receptor (alpha 1-AR) agonist phenylephrine (PE). PE stimulated the activity of p70S6K > 3-fold, and this increase was blocked by rapamycin, an immunosuppressant macrolide that selectively inhibits p70S6K. When administered for 3 days, PE stimulated a 30% increase in total protein content, a 2-fold increase in the incorporation of [14C]phenylalanine (14C-Phe) into protein, and a 50% increase in two-dimensional myocyte area. Rapamycin pretreatment (> or = 500 pg/mL) significantly inhibited each of these PE-stimulated changes. Two days of PE treatment resulted in a 1.6-fold increase in total RNA yield per dish, a 2-fold increase in incorporation of [14C]uridine into myocyte RNA, and increases in relative mRNA levels of the hypertrophy-associated atrial natriuretic factor (ANF, 2.1-fold) and skeletal alpha-actin (SK, 2.2-fold) genes. Although rapamycin abolished the PE-stimulated increases in total RNA and incorporation of [14C]uridine, it had no effect on the induction of the ANF and SK genes. LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3-K) activity, inhibited PE-stimulated increases in p70S6K activity and the incorporation of labeled precursors into myocyte protein and RNA. These results demonstrate that p70S6K is activated by the hypertrophic agent PE and that a PI3-K or PI3-K-like activity is required for p70S6K activation and myocyte hypertrophy. The data suggest that p70S6K activation may be required for PE-stimulated hypertrophy of cardiac myocytes. Our results demonstrate that intracellular signaling pathways responsible for transcriptional and translational responses diverge early after alpha 1-AR stimulation in cardiac myocytes.

The inhibition of vascular smooth muscle cell migration by peptide and antibody antagonists of the alphavbeta3 integrin complex is reversed by activated calcium/calmodulin- dependent protein kinase II.

The migration of vascular smooth muscle cells (VSMCs) is thought to play a key role in the pathogenesis of many vascular diseases and is regulated by soluble growth factors/ chemoattractants as well as interactions with the extracellular matrix. We have studied the effects of antibodies to rat beta3 and human alphavbeta3 integrins on the migration of VSMCs. Both integrin antibodies as well as cyclic RGD peptides that bind to the vitronectin receptors alphavbeta3 and alphavbeta5 significantly inhibited PDGF-directed migration. This resulted in a reduction in the accumulation of inositol (1,4,5) trisphosphate and the activation of calcium/calmodulin-dependent protein kinase II (CamKII), an important regulatory event in VSMC migration identified previously. PDGF-directed VSMC migration in the presence of the anti-integrin antibodies and cyclic RGD peptides was restored when intracellular CamKII activity was elevated by either raising intracellular calcium levels with the ionophore, ionomycin, or infecting with a replication-defective recombinant adenovirus expressing a constitutively activated CamKII cDNA (AdCMV.CKIID3). Rescue of rat VSMCs was also observed in stably transfected cell lines expressing constitutively activated but not wild-type CamKII. These observations identify a key intermediate in the regulation of VSMC migration by outside-in signaling from the integrin alphavbeta3.