Common carotid intima-media thickness does not add to Framingham risk score in individuals with diabetes mellitus: the USE-IMT initiative.

AIMS/HYPOTHESIS: The aim of this work was to investigate whether measurement of the mean common carotid intima-media thickness (CIMT) improves cardiovascular risk prediction in individuals with diabetes. METHODS: We performed a subanalysis among 4,220 individuals with diabetes in a large ongoing individual participant data meta-analysis involving 56,194 subjects from 17 population-based cohorts worldwide. We first refitted the risk factors of the Framingham heart risk score on the individuals without previous cardiovascular disease (baseline model) and then expanded this model with the mean common CIMT (CIMT model). The absolute 10 year risk for developing a myocardial infarction or stroke was estimated from both models. In individuals with diabetes we compared discrimination and calibration of the two models. Reclassification of individuals with diabetes was based on allocation to another cardiovascular risk category when mean common CIMT was added. RESULTS: During a median follow-up of 8.7 years, 684 first-time cardiovascular events occurred among the population with diabetes. The C statistic was 0.67 for the Framingham model and 0.68 for the CIMT model. The absolute 10 year risk for developing a myocardial infarction or stroke was 16% in both models. There was no net reclassification improvement with the addition of mean common CIMT (1.7%; 95% CI -1.8, 3.8). There were no differences in the results between men and women. CONCLUSIONS/INTERPRETATION: There is no improvement in risk prediction in individuals with diabetes when measurement of the mean common CIMT is added to the Framingham risk score. Therefore, this measurement is not recommended for improving individual cardiovascular risk stratification in individuals with diabetes.

Phospholemman does not participate in forskolin-induced swine carotid artery relaxation.

Phosphorylation of phospholemman (PLM) on ser68 has been proposed to at least partially mediate cyclic AMP (cAMP) mediated relaxation of arterial smooth muscle. We evaluated the time course of the phosphorylation of phospholemman (PLM) on ser68, myosin regulatory light chains (MRLC) on ser19, and heat shock protein 20 (HSP20) on ser16 during a transient forskolin-induced relaxation of histamine-stimulated swine carotid artery. We also evaluated the dose response for forskolin- and nitroglycerin-induced relaxation in phenylephrine-stimulated PLM-/- and PLM+/+ mice. The time course for changes in ser19 MRLC dephosphorylation and ser16 HSP20 phosphorylation was appropriate to explain the forskolin-induced relaxation and the recontraction observed upon washout of forskolin. However, the time course for changes in ser68 PLM phosphorylation was too slow to explain forskolin-induced changes in force. There was no difference in the phenylephrine contractile dose response or in forskolin-induced relaxation dose response observed in PLM-/- and PLM+/+ aortae. In aortae precontracted with phenylephrine, nitroglycerin induced a slightly, but significantly greater relaxation in PLM-/- compared to PLM+/+ aortae. These data are consistent with the hypothesis that ser19 MRLC dephosphorylation and ser16 HSP20 phosphorylation are involved in forskolin-induced relaxation. Our data suggest that PLM phosphorylation is not significantly involved in forskolin-induced arterial relaxation.

Magnesium relaxes arterial smooth muscle by decreasing intracellular Ca2+ without changing intracellular Mg2+.

Elevations in extracellular [Mg2+] ([Mg2+]o) relax vascular smooth muscle. We tested the hypothesis that elevated [Mg2+]o induces relaxation through reductions in myoplasmic [Ca2+] and myosin light chain phosphorylation without changing intracellular [Mg2+] ([Mg2+]i). Histamine stimulation of endothelium-free swine carotid medial tissues was associated with increases in both Fura 2- and aequorin-estimated myoplasmic [Ca2+], myosin phosphorylation, and force. Elevated [Mg2+]o decreased myoplasmic [Ca2+] and force to near resting values. However, elevated [Mg2+]o only transiently decreased myosin phosphorylation values: sustained [Mg2+]o-induced decreases in myoplasmic [Ca2+] and force were associated with inappropriately high myosin phosphorylation values. The elevated myosin phosphorylation during [Mg2+]o-induced relaxation was entirely on serine 19, the Ca2+/calmodulin-dependent myosin light chain kinase substrate. Myoplasmic [Mg2+] (estimated with Mag-Fura 2) did not significantly increase with elevated [Mg2+]o. These results are consistent with the hypothesis that increased [Mg2+]o induces relaxation by decreasing myoplasmic [Ca2+] without changing [Mg2+]i. These data also demonstrate dissociation of myosin phosphorylation from myoplasmic [Ca2+] and force during Mg(2+)-induced relaxation. This finding suggests the presence of a phosphorylation-independent (yet potentially Ca(2+)-dependent) mechanism for regulation of force in vascular smooth muscle.

Caldesmon and heat shock protein 20 phosphorylation in nitroglycerin- and magnesium-induced relaxation of swine carotid artery.

Nitrovasodilators, high extracellular Mg(2+), and some other relaxing agents can cause smooth muscle relaxation without reductions in myosin regulatory light chain (MRLC) phosphorylation. Relaxations without MRLC dephosphorylation suggest that other regulatory systems, beyond MRLC phosphorylation, are present in smooth muscle. We tested whether changes in caldesmon phosphorylation, heat shock protein 20 (HSP20) phosphorylation, or intracellular pH (pH(i)) could be responsible for relaxation without MRLC dephosphorylation. In unstimulated tissues, caldesmon was phosphorylated 1.02+/-0.10 mol P(i)/mol caldesmon (mean+/-1 S.E.M.), HSP20 was phosphorylated 0.005+/-0.003 mol P(i)/mol HSP20, and estimated pH(i) was 7.21+/-0.07. Histamine stimulation induced a contraction, an intracellular acidosis, but did not significantly change caldesmon or HSP20 phosphorylation. Addition of nitroglycerin induced a relaxation, significantly increased HSP20 phosphorylation to 0.18+/-0.02 mol P(i)/mol HSP20, did not significantly change caldesmon phosphorylation, and pH(i) returned to near unstimulated values. Increase in extracellular Mg(2+) to 10 mM induced a relaxation, but did not significantly change HSP20 or caldesmon phosphorylation. These data suggest that changes in caldesmon phosphorylation, HSP20 phosphorylation, or pH(i) cannot be the sole explanation for relaxation without MRLC dephosphorylation. However, it is possible that HSP20 phosphorylation may be involved in nitroglycerin-induced relaxation without MRLC dephosphorylation.

Myosin light chain kinase phosphorylation in nitrovasodilator induced swine carotid artery relaxation.

Nitrovasodilators are hypothesized to induce smooth muscle relaxation by their metabolism to nitric oxide, which then activates soluble guanylyl cyclase, increases [cGMP], and activates cGMP-dependent protein kinase. cGMP-dependent phosphorylation is then proposed to decrease intracellular [Ca2+] ([Ca2+]i) and to reduce the Ca(2+)-sensitivity of contraction. We hypothesized that one component of decreased Ca(2+)-sensitivity, reduced Ca(2+)-sensitivity of MLC phosphorylation, was due to phosphorylation of myosin light chain kinase (MLCK) on the peptide site A. In the swine carotid artery, histamine (10 microM) stimulation increased aequorin-estimated [Ca2+]i, MLCK site A phosphorylation, MLC phosphorylation, and force. Subsequent addition of 100 microM nitroglycerin (NTG) or 100 microM sodium nitroprusside (NP) to histamine-stimulated tissues increased [cGMP], decreased both MLC phosphorylation and force, but did not significantly alter [cAMP], [Ca2+]i, or MLCK site A phosphorylation. Addition of NTG and NP alone to unstimulated tissues increased MLCK site A phosphorylation, but did not alter [Ca2+]i. In tissues preincubated with NP, subsequent histamine contraction was slowed compared with controls, however, this slowed rate of contraction appeared to result from an attenuation of histamine-dependent increases in [Ca2+]i. These data suggest that, in swine carotid artery, nitrovasodilators can decrease the Ca(2+)-sensitivity of MLC phosphorylation without increasing MLCK site A phosphorylation. Nitrovasodilators, per se, can induce site A MLCK phosphorylation, potentially by cGMP dependent activation of cAMP-dependent protein kinase.

Measurement of changes in sarcoplasmic reticulum [Ca2+] in rat tail artery with targeted apoaequorin delivered by an adenoviral vector.

The physiologic relevance of Ca2+ release from the sarcoplasmic reticulum in arterial smooth muscle contraction is controversial. Therefore, we sought to measure changes in sarcoplasmic reticulum free [Ca2+] (i.e. [Ca2+]sr) in the intact rat tail artery. We exploited a novel technique to measure [Ca2+]sr with genetically targeted apoaequorin acting as a pseudo-luciferase rather than as classic aequorin. Intact rat tail arteries were infected with a replication deficient adenoviral vector (RAdER) containing the apoaequorin gene targeted to the sarcoplasmic reticulum. Addition of apoaequorin's substrate, coelenterazine, to the perfusate increased light production in a [Ca2+] dependent manner, consistent with apoaequorin action on coelenterazine. Within the limits of the photon counting system, imaging of infected rat tail artery segments revealed light production from the whole thickness of the vascular wall. Phenylephrine stimulation decreased apoaequorin generated light and induced a contraction. Washout of phenylephrine relaxed the tissues and increased light indicating refilling of the sarcoplasmic reticulum with Ca2+. Incubation in 10 microM cyclopiazonic acid, a SERCA inhibitor, did not alter apoaequorin generated light or induce a contraction. In the presence of cyclopiazonic acid, phenylephrine contractions were enhanced and apoaequorin generated light decreased further than that observed in the absence of cyclopiazonic acid. Cyclopiazonic acid also prevented the increase in apoaequorin generated light upon washout of phenylephrine, consistent with its inhibition of sarcoplasmic reticulum refilling. These results suggest that light production from targeted apoaequorin, delivered by a replication deficient adenovirus, is a valid measure of changes in [Ca2+]sr in the intact arterial wall. There appeared to be a correlation between Ca2+ release and contraction in these lightly loaded arteries.

Focal increases in [Ca2+]i may account for apparent low Ca2+ sensitivity in swine carotid artery.

Estimates of [Ca2+]i sensitivity in intact smooth muscle are frequently obtained by measuring [Ca2+]i with indicators such as aequorin or Fura-2. We investigated whether focal increases in [Ca2+]i could impair such measures of [Ca2+]i sensitivity. Stimulation of swine carotid artery with 10 microM histamine increased aequorin estimated [Ca2+]i, Fura-2 estimated [Ca2+]i and Ca2+ sensitivity without significantly altering the aequorin/Fura-2 ratio (an estimate of [Ca2+]i homogeneity). Subsequent inhibition of Na+/Ca2+ exchange by replacement of Na+ in the PSS with choline+ significantly increased aequorin-estimated [Ca2+]i but only minimally increased Fura-2 estimated [Ca2+]i, myosin light chain (MLC) phosphorylation and force. This resulted in a large increase in the aequorin/Fura-2 ratio, suggesting an increase in [Ca2+] inhomogeneity. Addition of 100 microM histamine to tissues in the choline+ buffer initially increased both aequorin and Fura-2 estimated [Ca2+]i, but after 10 min exposure both of the [Ca2+]i estimates declined to pre-histamine levels. Histamine addition significantly increased MLC phosphorylation and force, indicating increased Ca2+ sensitivity, but the aequorin/Fura-2 ratio remained elevated and unchanged from pre-histamine values. These data show that under certain conditions, aequorin and Fura-2 can yield widely differing estimates of [Ca2+]i and thus can cause misleading assessments of Ca2+ sensitization mechanisms. These discrepancies may arise from inhomogeneous or focal increases in [Ca2+]i which can be evaluated with the aequorin/Fura-2 ratio.

Current evidence suggests independent regulation of nuclear calcium.

We review and present current evidence supporting independent regulation of nuclear Ca2+ ([Ca2+]n). The nucleus and nuclear envelope contain proteins to both regulate and respond to changes in [Ca2+]n. However, this does not prove that [Ca2+]n is independently regulated from cytosolic Ca2+ ([Ca2+]c). Studies using fluorescent dyes suggested that changes in [Ca2+]n differed in magnitude from changes in [Ca2+]c. These studies have been criticised as the nuclear environment alters the fluorescent characteristics of these dyes. We have evaluated this question with aequorin targeted to the nucleus and cytoplasm and shown that the characteristics of the indicators are not altered in their respective environments. We have demonstrated that different stimuli induce changes in [Ca2+]n and [Ca2+]c that vary both temporally and in magnitude. The nucleus appeared to be shielded from increases in [Ca2+]c, either through a mechanism involving the nuclear envelope or by cytosolic buffering of localised increases in Ca2+. In addition, agonist stimulation resulted in an increase in [Ca2+]n, consistent with release from the perinuclear Ca2+ store. There was a stimulus dependence of the relation between [Ca2+]n and [Ca2+]c suggesting differential regulation of [Ca2+]n. These results have important implications for the role of Ca2+ as a specific regulator of nuclear events through Ca2+ binding proteins. In addition, they highlight the advantages of using targeted aequorin in intact cells to monitor changes in organelle [Ca2+].

Regulation of contraction and relaxation in arterial smooth muscle.

Intracellular calcium concentration ([Ca2+]i)-dependent activation of myosin light chain kinase and its phosphorylation of the 20-kd light chain of myosin is generally considered the primary mechanism responsible for regulation of contractile force in arterial smooth muscle. However, recent data suggest that the relation between [Ca2+]i and myosin light chain phosphorylation is variable and depends on the form of stimulation. The dependence of myosin phosphorylation on [Ca2+]i has been termed the "[Ca2+]i sensitivity of phosphorylation." The [Ca2+]i sensitivity of phosphorylation is "high" when relatively small increases in [Ca2+]i induce a large increase in myosin phosphorylation. Conversely, the [Ca2+]i sensitivity of phosphorylation is "low" when relatively large increases in [Ca2+]i are required to induce a small increase in myosin phosphorylation. There are two proposed mechanisms for changes in the [Ca2+]i sensitivity of phosphorylation: Ca(2+)-dependent decreases in the [Ca2+]i sensitivity of phosphorylation induced by phosphorylation of myosin light chain kinase by Ca(2+)-calmodulin protein kinase II and agonist-dependent increases in the [Ca2+]i sensitivity of phosphorylation by inhibition of a myosin light chain phosphatase. I will review the proposed mechanisms responsible for the regulation of [Ca2+]i and the [Ca2+]i sensitivity of phosphorylation in arterial smooth muscle.

pHi, [Ca2+]i, and myosin phosphorylation in histamine- and NH4(+)-induced swine carotid artery contraction.

We examined the interaction among changes in pHi, [Ca2+]i, myosin light-chain phosphorylation, and contraction in arterial smooth muscle stimulated by histamine, NH4+, Tris+, and/or changes in extracellular pH (pHo). We loaded swine carotid medial tissues with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein to measure pHi or aequorin to measure [Ca2+]i. Incubation of tissues in NH4+ increased pHi, [Ca2+]i, myosin phosphorylation, and force. Washout of NH4+ decreased pHi and transiently further increased in [Ca2+]i and force. Incubation of tissues in a similar concentration of Tris+ or increasing pHo also increased pHi; however, there were only modest changes in [Ca2+]i and force. Increasing extracellular pH coincidentally with washout of NH4+ prevented the decrease in pHi but did not affect the NH4+ washout-induced contraction. These data suggest that NH4+ altered [Ca2+]i and contraction by mechanisms other than its effects on pHi. The type of pH buffer did not affect the [Ca2+]i, myosin phosphorylation, or stress response to histamine stimulation. The time course of changes in pHi was much slower than the time course of histamine-induced changes in [Ca2+]i, myosin phosphorylation, and stress. Addition of 10 mmol/L NH4+ concurrently with histamine aborted the histamine-induced decrease in pHi and significantly slowed the histamine-induced increase in [Ca2+]i, myosin phosphorylation, and stress. There was little effect on histamine-induced increases in [Ca2+]i, myosin phosphorylation, or contraction when three other protocols aborted the histamine-induced decrease in pHi. These data show that incubation in NH4+ can alter [Ca2+]i and contraction in both unstimulated and histamine-stimulated smooth muscle. However, these effects were not caused by NH4(+)-dependent changes in pHi.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanisms responsible for forskolin-induced relaxation of rat tail artery.

The goal of the present study was to determine the physiologically relevant mechanisms for forskolin-induced relaxation of intact rat tail artery. We stimulated deendothelialized rat tail artery with phenylephrine and then relaxed the tissue with the addition of forskolin, a specific activator of adenylyl cyclase. We measured membrane potential with the use of microelectrodes, estimated intracellular Ca2+ concentration ([Ca2+]i) with the use of fura 2, and measured isometric force with a strain-gauge transducer. We found that 0.3 to 1.0 micromol/L forskolin relaxed 0.3 to 1.0 micromol/L phenylephrine-stimulated rat tail artery by decreasing the [Ca2+]i sensitivity of force as well as through repolarization. There was no evidence for forskolin-induced inhibition of Ca2+ influx beyond that associated with repolarization. There also was no evidence for forskolin-induced enhancement of Ca2+ efflux or sequestration. Inhibition of ATP-activated K+ channels with 10 micromol/L glibenclamide, Ca2+-activated K+ channels with 50 nmol/L iberiotoxin, Ca2+-activated K+ channels with 3 or 10 mmol/L tetraethylammonium ion, inwardly rectified K+ channels with 20 micromol/L Ba2+, and voltage-activated K+ channels with 0.5 mmol/L 4-aminopyridine did not significantly attenuate forskolin-induced reductions in [Ca2+]i or force. Forskolin-induced repolarization was not altered by 10 micromol/L glibenclamide or 0.5 mmol/L 4-aminopyridine. These data suggest that these K+ channels were not individually involved in forskolin-induced relaxation and that other channels and/or multiple channels are involved in forskolin-induced repolarization of intact rat tail artery. Our data also suggest that forskolin-induced relaxation of intact rat tail artery occurred primarily through repolarization and reductions in the [Ca2+]i sensitivity of force.

[Ca2+], not diacylglycerol, is the primary regulator of sustained swine arterial smooth muscle contraction.

Sustained smooth muscle contraction has been proposed to be regulated by either 1) sustained increases in intracellular Ca2+ concentration [(Ca2+]i)-dependent myosin phosphorylation or 2) diacylglycerol-dependent protein kinase C activation. We measured diacylglycerol mass with the diacylglycerol kinase assay and myoplasmic [Ca2+] with aequorin in swine carotid medial smooth muscle. Sustained and significant increases in [Ca2+], myosin light chain phosphorylation, and isometric stress were observed with histamine or endothelin stimulation. Neither stimuli, however, induced significant increases in diacylglycerol mass. Relaxation of histamine-stimulated tissues was induced by removal of histamine or removal of extracellular CaCl2 in the continued presence of histamine. The rate of decline of both [Ca2+] and force was similar in both protocols, suggesting that removal of Ca2+ (without removing the stimulus) was equivalent to removal of the stimulus. These data suggest that [Ca2+]i is the primary regulator of sustained swine arterial smooth muscle contraction, whereas diacylglycerol has, at most, only a minor role.

Na(+)-Ca2+ exchange, myoplasmic Ca2+ concentration, and contraction of arterial smooth muscle.

Na(+)-Ca2+ exchange is proposed to be an important regulator of myoplasmic intracellular Ca2+ concentration ([Ca2+]i) and contraction in vascular smooth muscle. We investigated the role of Na(+)-Ca2+ exchange in regulating [Ca2+]i in swine carotid arterial tissues that were loaded with aequorin to allow simultaneous measurement of [Ca2+]i and force. Reversal of Na(+)-Ca2+ exchange, by reduction of extracellular Na+ concentration ([Na+]o) to 1.2 mM, induced a large increase in aequorin-estimated [Ca2+]i and a low [Ca2+]i sensitivity. The contraction induced by 1.2 mM [Na+]o was partially caused by depolarization and opening of L-type Ca2+ channels because 10 microM diltiazem partially attenuated the 1.2 mM [Na+]o-induced increases in [Ca2+]i. High dose ouabain (10 microM), a putative endogenous Na+,K(+)-ATPase inhibitor, increased both [Ca2+]i and force. However, the increases in [Ca2+]i and force were mostly blocked by 10 microM phentolamine, suggesting the predominant effect of ouabain was to increase norepinephrine release from nerve terminals. In the presence of 10 microM phentolamine, 10 microM ouabain slightly accentuated 1 microM histamine-induced increases in [Ca2+]i and force. The ouabain dose necessary to induce contraction in the absence of phentolamine was significantly less than the ouabain dose necessary to accentuate histamine-induced contractions in the presence of phentolamine. These results suggest that Na(+)-Ca2+ exchange exists in swine arterial smooth muscle. These data also suggest that ouabain (which should increase [Na+]i and inhibit Na(+)-Ca2+ exchange) primarily enhances contractile function in the swine carotid artery by releasing catecholamines from nerve terminals; direct action of Na+,K(+)-ATPase inhibitors on smooth muscle appears to occur only with very high doses.

Metformin relaxes rat tail artery by repolarization and resultant decreases in Ca2+ influx and intracellular [Ca2+].

BACKGROUND: Metformin treatment of type II diabetes is frequently associated with decreases in blood pressure, an effect that could result from a direct action of metformin on arterial smooth muscle. OBJECTIVE: To determine the mechanisms responsible for arterial smooth muscle relaxation induced by acute application of metformin and to evaluate the effect of insulin pretreatment on intracellular [Ca2+] ([Ca2+]i) and contraction in an intact artery. METHODS: We stimulated intact deendothelialized rat tail artery with phenylephrine, relaxed the tissue by adding increasing concentrations of metformin, and measured the membrane potential (Em), Mn2+ influx, Fura 2-estimated [Ca2+]i, and isometric force. We also evaluated the effect of insulin pretreatment on aequorin-estimated [Ca2+]i in deendothelialized swine carotid artery. RESULTS: In rat tail artery we found that a high concentration of metformin-induced repolarization associated with proportional decreases in Mn2+ influx, Fura 2-estimated [Ca2+]i, and isometric force. Incubation of swine carotid artery in 100 mU/ml insulin for 30 min or overnight (16-22 h) did not significantly alter histamine or high-K+-induced increases in [Ca2+]i or contraction. CONCLUSION: These data suggest that acute administration of high concentrations of metformin induces rat tail artery relaxation primarily by repolarization. Additionally, we found that insulin was not vasoactive in the swine carotid artery. It is possible that insulin may alter [Ca2+]i handling in other arteries, in other species, or only in cultured smooth muscle.

Growth of the human heart relative to body surface area.

To determine the growth rate of the human heart in relation to body surface area, normal data obtained by echocardiography and angiocardiography were analyzed with respect to changes in body size occurring between infancy and adulthood. Published data were used to determine the following for 7 theoretical subjects ranging in size from 0.2 m2 (normal newborn) to 2.2 m2 (large adult): average left ventricular (LV) end-diastolic diameter; aortic root diameter; right pulmonary artery diameter; aortic valve area; LV end-diastolic volume; and LV mass. Linear dimensions (LV, aortic and right pulmonary artery diameter) increased as a square root function of surface area and could not be described by a constant "index" of dimension to surface area. Aortic valve area was linearly related to surface area and equalled 3.4 cm2/m2 for subjects of all sizes. LV volume and mass were exponentially related to surface area. This study indicates that intracardiac areas may be indexed for body surface area, but that linear dimensions and volumes have a nonlinear relation to surface area and are more appropriately indexed by surface area to the 0.5 and 1.5 power, respectively.

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.

Localization of heat shock protein 20 in swine carotid artery.

BACKGROUND: Cyclic nucleotides can relax vascular smooth muscle by mechanisms distal to myosin regulatory light chain (MRLC) phosphorylation. This mechanism, termed relaxation without MRLC dephosphorylation, may be regulated by ser16 phosphorylation of heat shock protein 20 (HSP20). RESULTS: Confocal imaging of HSP20 in smooth muscle tissues revealed that HSP20 was present throughout the cytoplasm, although some focal regions of the cytoplasm were found to contain more HSP20 than the remaining cytoplasm. The distribution of HSP20 within the cytoplasm was not altered by histamine, forskolin, or nitroglycerin. CONCLUSION: Cytoplasmic localization of HSP20 is consistent with a potential function of HSP20 as a regulator of smooth muscle contractile force.

Number needed to screen: development of a statistic for disease screening.

OBJECTIVES: To develop the number needed to screen, a new statistic to overcome inappropriate national strategies for disease screening. Number needed to screen is defined as the number of people that need to be screened for a given duration to prevent one death or adverse event. DESIGN: Number needed to screen was calculated from clinical trials that directly measured the effect of a screening strategy. From clinical trials that measured treatment benefit, the number needed to screen was estimated as the number needed to treat from the trial divided by the prevalence of heretofore unrecognised or untreated disease. Directly calculated values were then compared with estimate number needed to screen values. SUBJECTS: Standard literature review. RESULTS: For prevention of total mortality the most effective screening test was a lipid profile. The estimated number needed to screen for dyslipidaemia (low density lipoprotein cholesterol concentration >4.14 mmol/1) was 418 if detection was followed by pravastatin treatment for 5 years. This indicates that one death in 5 years could be prevented by screening 418 people. The estimated number needed to screen for hypertension was between 274 and 1307 for 5 years (for 10 mm Hg and 6 mm Hg diastolic blood pressure reduction respectively) if detection was followed by treatment based on a diuretic. Screening with haemoccult testing and mammography significantly decreased cancer specific, but not total, mortality. The number needed to screen for haemoccult screening to prevent a death from colon cancer was 1374 for 5 years, and the number needed to screen for mammography to prevent a death from breast cancer was 2451 for 5 years for women aged 50-59. CONCLUSION: These data allow the clinician to prioritise screening strategies. Of the screening strategies evaluated, screening for, and treatment of, dyslipidaemia and hypertension seem to produce the largest clinical benefit.

Number-needed-to-treat analysis of the prevention of myocardial infarction and death by antidyslipidemic therapy.

BACKGROUND: Atherosclerosis of the coronary arteries is the most common cause of death in the United States for persons over the age of 45. Dyslipidemia is one of the risk factors for the development of coronary atherosclerosis. Recent studies suggest that treating dyslipidemia in persons with coronary atherosclerosis may decrease morbidity and mortality. METHODS: A meta-analysis of 33 studies on the clinical and angiographic benefits of treating dyslipidemia in the prevention of morbidity and mortality from cardiovascular disease was performed. These benefits are quantitated in the form of "number needed to treat" (NNT) as an estimate of the public health benefit. The NNT is defined as the number of people that need to be treated to prevent one event. RESULTS: Treatment of dyslipidemia in persons with multiple atherosclerosis risk factors alone, ie, primary prevention, was effective in preventing myocardial infarction and all-cause death. In six trials of primary prevention, excluding the British cooperative trial using clofibrate, the NNT was 53 to prevent a nonfatal MI and 190 to prevent all-cause death (4.8 years treatment with total cholesterol reduction of 15%). Treatment of dyslipidemia in people with known atherosclerosis, ie, secondary and tertiary prevention, was also effective in preventing myocardial infarctions and death from all causes. For 23 trials of secondary and tertiary prevention, the NNT was 37 to prevent death from any cause (4.9 years treatment with total cholesterol reduction of 18%). In the trials with quantitative angiography, the NNT was 7 to prevent progression of coronary atherosclerosis and 10 to induce regression of coronary atherosclerosis (2.5 years treatment with a low-density lipoprotein cholesterol reduction of 28%). Similar benefits were observed in those trials employing HMG CoA reductase inhibitors. Benefits may be similar with niacin or dietary therapy, but these therapies did not reach significance in all categories of benefits, potentially due to beta error. These treatment benefits are comparable to other secondary prevention measures such as aspirin or beta blockers. The benefits appeared to extend to persons over 65, with less clearly defined benefits for women. CONCLUSIONS: These results support the overall clinical benefit of treating dyslipidemia, both in persons with and without known atherosclerosis.

Does pseudoephedrine increase blood pressure in patients with controlled hypertension?

BACKGROUND: The use of the decongestant pseudoephedrine has been avoided in hypertensive patients with little evidence to support this caution. The purpose of this study was to determine the effects of therapeutic doses of pseudoephedrine on blood pressure in patients with controlled hypertension. METHODS: Twenty-five patients with controlled hypertension were enrolled in a randomized, placebo-controlled, double-blind crossover trial. This was a 4-week study in which all participants received placebo during the 1st and 3rd weeks. Subjects were randomly assigned to receive pseudoephedrine or placebo during the 2nd week of the study, and to receive the opposite during the 4th week of the study. Blood pressure readings were taken at the beginning and end of each week of the trial, and at a 1-week poststudy follow-up visit. RESULTS: The mean systolic pressure was 133 mm Hg in patients receiving both pseudoephedrine and placebo (P = NS). The mean diastolic pressure was 82 mm Hg in patients receiving pseudoephedrine and 82.5 mm Hg in patients receiving placebo. Mean pulse rates were 76.8 and 75.2 beats per minute in patients given pseudoephedrine and those given placebo, respectively (P = NS). There were no statistically significant or clinically important differences in the mean systolic or mean diastolic blood pressures among the groups during the entire 4-week course of the study. CONCLUSIONS: At standard doses, pseudoephedrine has no significant effect on systolic or diastolic blood pressure in patients with controlled hypertension.