Beta-arrestins regulate atherosclerosis and neointimal hyperplasia by controlling smooth muscle cell proliferation and migration.

Atherosclerosis and arterial injury-induced neointimal hyperplasia involve medial smooth muscle cell (SMC) proliferation and migration into the arterial intima. Because many 7-transmembrane and growth factor receptors promote atherosclerosis, we hypothesized that the multifunctional adaptor proteins beta-arrestin1 and -2 might regulate this pathological process. Deficiency of beta-arrestin2 in ldlr(-/-) mice reduced aortic atherosclerosis by 40% and decreased the prevalence of atheroma SMCs by 35%, suggesting that beta-arrestin2 promotes atherosclerosis through effects on SMCs. To test this potential atherogenic mechanism more specifically, we performed carotid endothelial denudation in congenic wild-type, beta-arrestin1(-/-), and beta-arrestin2(-/-) mice. Neointimal hyperplasia was enhanced in beta-arrestin1(-/-) mice, and diminished in beta-arrestin2(-/-) mice. Neointimal cells expressed SMC markers and did not derive from bone marrow progenitors, as demonstrated by bone marrow transplantation with green fluorescent protein-transgenic cells. Moreover, the reduction in neointimal hyperplasia seen in beta-arrestin2(-/-) mice was not altered by transplantation with either wild-type or beta-arrestin2(-/-) bone marrow cells. After carotid injury, medial SMC extracellular signal-regulated kinase activation and proliferation were increased in beta-arrestin1(-/-) and decreased in beta-arrestin2(-/-) mice. Concordantly, thymidine incorporation and extracellular signal-regulated kinase activation and migration evoked by 7-transmembrane receptors were greater than wild type in beta-arrestin1(-/-) SMCs and less in beta-arrestin2(-/-) SMCs. Proliferation was less than wild type in beta-arrestin2(-/-) SMCs but not in beta-arrestin2(-/-) endothelial cells. We conclude that beta-arrestin2 aggravates atherosclerosis through mechanisms involving SMC proliferation and migration and that these SMC activities are regulated reciprocally by beta-arrestin2 and beta-arrestin1. These findings identify inhibition of beta-arrestin2 as a novel therapeutic strategy for combating atherosclerosis and arterial restenosis after angioplasty.

Experimental Studies of the Coherence of Microstructure-Fiber Supercontinuum

The phase coherence of supercontinuum generation in microstructure fiber is quantified by performing a Young's type interference experiment between independently generated supercontinua from two separate fiber segments. Analysis of the resulting interferogram yields the wavelength dependence of the magnitude of the mutual degree of coherence, and a comparison of experimental results with numerical simulations suggests that the primary source of coherence degradation is the technical noise-induced fluctuations in the injected peak power. © 2003 Optical Society of America.

Diabetes-induced activation of protein kinase C inhibits store-operated Ca2+ uptake in rat retinal microvascular smooth muscle.

AIMS/HYPOTHESIS: To assess the effects of diabetes-induced activation of protein kinase C (PKC) on voltage-dependent and voltage-independent Ca2+ influx pathways in retinal microvascular smooth muscle cells. METHODS: Cytosolic Ca2+ was estimated in freshly isolated rat retinal arterioles from streptozotocin-induced diabetic and non-diabetic rats using fura-2 microfluorimetry. Voltage-dependent Ca2+ influx was tested by measuring rises in [Ca2+]i with KCl (100 mmol/l) and store-operated Ca2+ influx was assessed by depleting [Ca2+]i stores with Ca2+ free medium containing 5 micromol/l cyclopiazonic acid over 10 min and subsequently measuring the rate of rise in Ca2+ on adding 2 mmol/l or 10 mmol/l Ca2+ solution. RESULTS: Ca2+ entry through voltage-dependent L-type Ca2+ channels was unaffected by diabetes. In contrast, store-operated Ca2+ influx was attenuated. In microvessels from non-diabetic rats 20 mmol/l D-mannitol had no effect on store-operated Ca2+ influx. Diabetic rats injected daily with insulin had store-operated Ca2+ influx rates similar to non-diabetic control rats. The reduced Ca2+ entry in diabetic microvessels was reversed by 2-h exposure to 100 nmol/l staurosporine, a non-specific PKC antagonist and was mimicked in microvessels from non-diabetic rats by 10-min exposure to the PKC activator phorbol myristate acetate (100 nmol/l). The specific PKCbeta antagonist LY379196 (100 nmol/l) also reversed the poor Ca2+ influx although its action was less efficacious than staurosporine. CONCLUSION/INTERPRETATION: These results show that store-operated Ca2+ influx is inhibited in retinal arterioles from rats having sustained increased blood glucose and that PKCbeta seems to play a role in mediating this effect.

Glucocorticoid induced muscle atrophy is associated with upregulation of myostatin gene expression

The mechanisms by which excessive glucocorticoids cause muscular atrophy remain unclear. We previously demonstrated that dexamethasone increases the expression of myostatin, a negative regulator of skeletal muscle mass, in vitro. In the present study, we tested the hypothesis that dexamethasone-induced muscle loss is associated with increased myostatin expression in vivo. Daily administration (60, 600, 1,200 micro g/kg body wt) of dexamethasone for 5 days resulted in rapid, dose-dependent loss of body weight (-4.0, -13.4, -17.2%, respectively, P <0.05 for each comparison), and muscle atrophy (6.3, 15.0, 16.6% below controls, respectively). These changes were associated with dose-dependent, marked induction of intramuscular myostatin mRNA (66.3, 450, 527.6% increase above controls, P <0.05 for each comparison) and protein expression (0.0, 260.5, 318.4% increase above controls, P <0.05). We found that the effect of dexamethasone on body weight and muscle loss and upregulation of intramuscular myostatin expression was time dependent. When dexamethasone treatment (600 micro g. kg-1. day-1) was extended from 5 to 10 days, the rate of body weight loss was markedly reduced to approximately 2% within this extended period. The concentrations of intramuscular myosin heavy chain type II in dexamethasone-treated rats were significantly lower (-43% after 5-day treatment, -14% after 10-day treatment) than their respective corresponding controls. The intramuscular myostatin concentration in rats treated with dexamethasone for 10 days returned to basal level. Concurrent treatment with RU-486 blocked dexamethasone-induced myostatin expression and significantly attenuated body loss and muscle atrophy. We propose that dexamethasone-induced muscle loss is mediated, at least in part, by the upregulation of myostatin expression through a glucocorticoid receptor-mediated pathway.

Ca(2+)-activated Cl(-) current in sheep lymphatic smooth muscle.

Freshly dispersed sheep mesenteric lymphatic smooth muscle cells were studied at 37 degrees C using the perforated patch-clamp technique with Cs(+)- and K(+)-filled pipettes. Depolarizing steps evoked currents that consisted of L-type Ca(2+) [I(Ca(L))] current and a slowly developing current. The slow current reversed at 1 +/- 1.5 mV with symmetrical Cl(-) concentrations compared with 23.2 +/- 1.2 mV (n = 5) and -34.3 +/- 3.5 mV (n = 4) when external Cl(-) was substituted with either glutamate (86 mM) or I(-) (125 mM). Nifedipine (1 microM) blocked and BAY K 8644 enhanced I(Ca(L)), the slow-developing sustained current, and the tail current. The Cl(-) channel blocker anthracene-9-carboxylic acid (9-AC) reduced only the slowly developing inward and tail currents. Application of caffeine (10 mM) to voltage-clamped cells evoked currents that reversed close to the Cl(-) equilibrium potential and were sensitive to 9-AC. Small spontaneous transient depolarizations and larger action potentials were observed in current clamp, and these were blocked by 9-AC. Evoked action potentials were triphasic and had a prominent plateau phase that was selectively blocked by 9-AC. Similarly, fluid output was reduced by 9-AC in doubly cannulated segments of spontaneously pumping sheep lymphatics, suggesting that the Ca(2+)-activated Cl(-) current plays an important role in the electrical activity underlying spontaneous activity in this tissue. PMID: 11029279 [PubMed - indexed for MEDLINE]

Ca2+ current and Ca(2+)-activated chloride current in isolated smooth muscle cells of the sheep urethra.

1. Isolated sheep urethral cells were studied using the perforated patch clamp technique (T = 37 degrees C). Depolarizing steps ranging from -40 to -10 mV evoked an inward current that peaked within 10 ms and a slower inward current. Stepping back to the holding potential of -80 mV evoked large inward tail currents. All three currents were abolished by nifedipine (1 microM). Substitution of external Ca2+ with Ba2+ resulted in potentiation of the fast inward current and blockade of the slow current and tails. 2. Changing the chloride equilibrium potential (ECl) from 0 to +27 mV shifted the reversal potential of the tail currents from 1 +/- 1 to 27 +/- 1 mV (number of cells, n = 5). Chloride channel blockers, niflumic acid (10 microM) and anthracene-9-carboxylic acid (9AC, 1 mM), reduced the slow current and tails suggesting that these were Ca(2+)-activated Cl- currents, ICl(Ca). 4. Caffeine (10 mM) induced currents that reversed at ECl and were blocked by niflumic acid (10 microM). 5. In current clamp mode, some cells developed spontaneous transient depolarizations (STDs) and action potentials. Short exposure to nifedipine blocked the action potentials and unmasked STDs. In contrast, 9AC and niflumic acid reduced the amplitude of the STDs and blocked the action potentials. 6. In conclusion, these cells have both L-type ICa and ICl(Ca). The former appears to be responsible for the upstroke of the action potential, while the latter may act as a pacemaker current.

Evidence for two endothelin Et(A) receptor subtypes in rabbit arteriolar smooth muscle.

1. Effects of endothelin-1 (Et-1) were studied on membrane currents in choroidal arteriolar smooth muscle by using perforated patch-clamp recordings. 2. Et-1 (10 nM) activated oscillatory Ca(2+)-activated Cl(-)-currents (I(Cl(Ca))) which could not be reversed by washing out. 3. Currents through L-type Ca(2+) channels were resolved in a divalent free medium (I(Ca(L)Na)). Et-1 reduced I(Ca(L)Na) by 75 +/- 7% within 30 s and this effect faded over 5 min, when the depression remained constant. On washing out Et-1, I(Ca(L)Na) almost completely recovered within 10 s. 4. BQ123 (1 microM), a peptide Et(A) receptor blocker, prevented the activation of I(Cl(Ca)), but failed to inhibit I(Cl(Ca)) transients once they had been initiated. In contrast, BQ123 not only prevented but also reversed the inhibition of I(Ca(L)Na) by Et-1. BQ788 (1 microM), an Et(B) receptor antagonist, did not prevent the activation of I(Cl(Ca)) or the inhibition of I(Ca(L)Na) by Et-1. 5. ABT-627 (10 nM), a non-peptide Et(A) receptor antagonist also blocked the activation of I(Cl(Ca)). However, on I(Ca(L)Na), ABT-627 (10 nM) mimicked the action of Et-1 an effect blocked by BQ123 suggesting that ABT-627 acted as an agonist. 6. The data are consistent with choroidal arteriolar smooth muscle cells having two types of Et(A) receptor, one where BQ123 is an antagonist and ABT-627 an agonist, where ligands dissociate freely and this receptor is coupled to inhibition of L-type Ca(2+) channels. In the other, BQ123 and ABT-627 are both antagonists and with Et-1 the receptor converts to a high affinity state producing the classical irreversible activation I(Cl(Ca)).

The smooth muscle pharmacology of maximakinin, a receptor-selective, bradykinin-related nonadecapeptide from the venom of the Chinese toad, Bombina maxima.

Structural homologues of vertebrate regulatory peptides found in defensive skin secretions of anuran amphibians often display enhanced bioactivity and receptor binding when compared with endogenous mammalian peptide ligands. Maximakinin, a novel N-terminally extended bradykinin (DLPKINRKGPRPPGFSPFR) from the skin venom of a Chinese toad (Bombina maxima), displays such activity enhancement when compared with bradykinin but is additionally highly selective for mammalian arterial smooth muscle bradykinin receptors displaying a 50-fold increase in molar potency in this smooth muscle type. In contrast, a 100-fold decrease in molar potency was observed at bradykinin receptors in intestinal and uterine smooth muscle preparations. Maximakinin has thus evolved as a “smart” defensive weapon in the toad with receptor/tissue selective targeting. Natural selection of amphibian skin venom peptides for antipredator defence, through inter-species delivery by an exogenous secretory mode, produces subtle structural stabilisation modifications that can potentially provide new insights for the design of selectively targeted peptide therapeutics.

Cellular physiology of retinal and choroidal arteriolar smooth muscle cells.

Control of ocular blood flow occurs predominantly at the level of the retinal and choroidal arterioles. The present article provides an overview of the Ca2 + handling mechanisms and plasmalemmal ion channels involved in the regulation of retinal and choroidal arteriolar smooth muscle tone. Increases in global intracellular free Ca2 + ([Ca2 +]i) involve multiple mechanisms, including agonist-dependent release of Ca2 + from intracellular stores through activation of the inositol trisphosphate (IP3) pathway. Ca2 + enters by voltage-dependent L-type Ca2 + channels and novel dihydropyridine-sensitive store-operated nonselective cation channels. Ca2 + extrusion is mediated by plasmalemmal Ca2 +-ATPases and through Na+/Ca2+ exchange. Local Ca2 + transients (Ca2 + sparks) play an important excitatory role, acting as the building blocks for more global Ca2 + signals that can initiate vasoconstriction. K+ and Cl- channels may also affect cell function by modulating membrane potential. The precise contribution of each of these mechanisms to the regulation of retinal and choroidal perfusion in vivo warrants future investigation.

Iron abundances from optical FeIII absorption lines in B-type stellar spectra

The role of optical FeIII absorption lines in B-type stars as iron abundance diagnostics is considered. To date, ultraviolet Fe lines have been widely used in B-type stars, although line blending can severely hinder their diagnostic power. Using optical spectra, covering a wavelength range ~3560-9200Å, a sample of Galactic B-type main-sequence and supergiant stars of spectral types B0.5 to B7 are investigated. A comparison of the observed FeIII spectra of supergiants, and those predicted from the model atmosphere codes TLUSTY [plane-parallel, non-local thermodynamic equilibrium (LTE)], with spectra generated using SYNSPEC (LTE), and CMFGEN (spherical, non-LTE), reveal that non-LTE effects appear small. In addition, a sample of main-sequence and supergiant objects, observed with the Fiber-fed Extended Range Optical Spectrograph (FEROS), reveal LTE abundance estimates consistent with the Galactic environment and previous optical studies. Based on the present study, we list a number of FeIII transitions which we recommend for estimating the iron abundance from early B-type stellar spectra.

Characterization of T-type calcium current and its contribution to electrical activity in rabbit urethra.

Rabbit urethral smooth muscle cells were studied at 37 degrees C by using the amphotericin B perforated-patch configuration of the patch-clamp technique, using Cs(+)-rich pipette solutions. Two components of current, with electrophysiological and pharmacological properties typical of T- and L-type Ca(2+) currents, were recorded. Fitting steady-state inactivation curves for the L current with a Boltzmann equation yielded a V(1/2) of -41 +/- 3 mV. In contrast, the T current inactivated with a V(1/2) of -76 +/- 2 mV. The L currents were reduced by nifedipine (IC(50) = 225 +/- 84 nM), Ni(2+) (IC(50) = 324 +/- 74 microM), and mibefradil (IC(50) = 2.6 +/- 1.1 microM) but were enhanced when external Ca(2+) was substituted with Ba(2+). The T current was little affected by nifedipine at concentrations

Abnormal integrity of association fiber tracts in amnestic mild cognitive impairment.

The association fiber tracts integrity of the inter-hemispheric and within-hemispheric communication was poor understood in amnestic type mild cognitive impairment (aMCI) patients by diffusion tensor imaging (DTI). A region of interest-based DTI approach was applied to explore fiber tract differences between 22 aMCI patients and 22 well-matched normal aging. Correlations were also sought between fractional anisotropy (FA) values and the cognitive performance scores in the aMCI patients. Extensive impairment of association fiber tracts integrity was observed in aMCI patients, including bilateral inferior fronto-occipital fascicles, the genu of corpus callosum, bilateral cingulate bundles and bilateral superior longitudinal fascicles II (SLE II) subcomponent. In addition, the FA value of right SLE II was significantly negatively correlated to the performance of Trail Making Test A and B, whilst the values of right posterior cingulate bundle was significantly positive correlation with MMSE score. As aMCI is a putative prodromal syndrome to Alzheimer's disease (AD), this study suggested that investigation of association fiber tracts between remote cortexes may yield important new data to predict whether a patient will eventually develop AD.

Ca2+-activated Cl- current in retinal arteriolar smooth muscle.

PURPOSE: To characterize the biophysical, pharmacologic, and functional properties of the Ca(2+)-activated Cl(-) current in retinal arteriolar myocytes. METHODS: Whole-cell perforated patch-clamp recordings were made from myocytes within intact isolated arteriolar segments. Arteriolar tone was assessed using pressure myography. RESULTS: Depolarizing of voltage steps to -40 mV and greater activated an L-type Ca(2+) current (I(Ca(L))) that was followed by a sustained current. Large tail currents (I(tail)) were observed on stepping back to -80 mV. The sustained current and I(tail) reversed close to 0 mV in symmetrical Cl(-) concentrations. The ion selectivity sequence for I(tail) was I(-)> Cl(-)> glucuronate. Outward I(tail) was sensitive to the Cl(-) channel blockers 9-anthracene-carboxylic acid (9-AC; 1 mM), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS; 1 mM), and disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS; 1 mM), but only DIDS produced a substantial (78%) block of inward tail currents at -100 mV. I(tail) was decreased in magnitude when the normal bathing medium was substituted with Ca(2+)-free solution or if I(Ca(L)) was inhibited by 1 microM nimodipine. Caffeine (10 mM) produced large transient currents that reversed close to the Cl(-) equilibrium potential and were blocked by 1 mM DIDS or 100 microM tetracaine. DIDS had no effect on basal vascular tone in pressurized arterioles but dramatically reduced the level of vasoconstriction observed in the presence of 10 nM endothelin-1. CONCLUSIONS: Retinal arteriolar myocytes have I(Cl(Ca)), which may be activated by Ca(2+) entry through L-type Ca(2+) channels or Ca(2+) release from intracellular stores. This current appears to contribute to agonist-induced retinal vasoconstriction.

PdT-2: a novel myotropic Type-2 tryptophyllin from the skin secretion of the Mexican giant leaf frog, Pachymedusa dacnicolor.

Amphibian skin secretions represent a unique resource for the discovery of new bioactive peptides. Here we report the isolation, structural and functional characterization of a novel heptapeptide amide, DMSPPWHamide, from the defensive skin secretion of the Mexican giant leaf frog, Pachymedusa dacnicolor. This peptide is of unique primary structure and has been classified as a member of the rather heterogenous tryptophyllin-2 (T-2) family of amphibian skin peptides and named P. dacnicolor Tryptophyllin-2 (PdT-2) in accordance. PdT-2 is the first Type 2-tryptophyllin to possess discrete bioactivity. Both natural and synthetic replicates of the peptide were found to contract the smooth muscle of rat urinary bladder, the latter displaying an EC50 of 4 nM.

Rho kinase and protein kinase C involvement in vascular smooth muscle myofilament calcium sensitization in arteries from diabetic rats.

BACKGROUND AND PURPOSE: Diabetes mellitus (DM) causes multiple dysfunctions including circulatory disorders such as cardiomyopathy, angiopathy, atherosclerosis and arterial hypertension. Rho kinase (ROCK) and protein kinase C (PKC) regulate vascular smooth muscle (VSM) Ca(2+) sensitivity, thus enhancing VSM contraction, and up-regulation of both enzymes in DM is well known. We postulated that in DM, Ca(2+) sensitization occurs in diabetic arteries due to increased ROCK and/or PKC activity. EXPERIMENTAL APPROACH: Rats were rendered hyperglycaemic by i.p. injection of streptozotocin. Age-matched control tissues were used for comparison. Contractile responses to phenylephrine (Phe) and different Ca(2+) concentrations were recorded, respectively, from intact and chemically permeabilized vascular rings from aorta, tail and mesenteric arteries. KEY RESULTS: Diabetic tail and mesenteric arteries demonstrated markedly enhanced sensitivity to Phe while these changes were not observed in aorta. The ROCK inhibitor HA1077, but not the PKC inhibitor chelerythrine, caused significant reduction in sensitivity to agonist in diabetic vessels. Similar changes were observed for myofilament Ca(2+) sensitivity, which was again enhanced in DM in tail and mesenteric arteries, but not in aorta, and could be reduced by both the ROCK and PKC blockers. CONCLUSIONS AND IMPLICATIONS: We conclude that in DM enhanced myofilament Ca(2+) sensitivity is mainly manifested in muscular-type blood vessels and thus likely to contribute to the development of hypertension. Both PKC and, in particular, ROCK are involved in this phenomenon. This highlights their potential usefulness as drug targets in the pharmacological management of DM-associated vascular dysfunction.