Derivation of a tuberculosis screening rule for sub-Saharan African prisons.

SETTING: Lusaka Central Prison, Zambia. OBJECTIVE: To derive screening rules for tuberculosis (TB) using data collected during a prison-wide TB and human immunodeficiency virus (HIV) screening program. DESIGN: We derived rules with two methodologies: logistic regression and classification and regression trees (C&RT). We evaluated the performance of the derived rules as well as existing World Health Organization (WHO) screening recommendations in our cohort of inmates, as measured by sensitivity, specificity, and positive and negative predictive values. RESULTS: The C&RT-derived rule recommended diagnostic testing of all inmates who were underweight (defined as body mass index [BMI] < 18.5 kg/m(2)] or HIV-infected; the C&RT-derived rule had 60% sensitivity and 71% specificity. The logistic regression-derived rule recommended diagnostic testing of inmates who were underweight, HIV-infected or had chest pain; the logistic regression-derived rule had 74% sensitivity and 57% specificity. Two of the WHO recommendations had sensitivities that were similar to our logistic regression rule but had poorer specificities, resulting in a greater testing burden. CONCLUSION: Low BMI and HIV infection were the most robust predictors of TB in our inmates; chest pain was additionally retained in one model. BMI and HIV should be further evaluated as the basis for TB screening rules for inmates, with modification as needed to improve the performance of the rules.

Relationships between metrics of visit-to-visit variability of blood pressure.

This paper examines relationships between metrics of visit-to-visit variability (VVV) of blood pressure (BP) to determine which metrics should be calculated in studies of the association of VVV with health outcomes. We examined correlation and agreement between quintiles for standard deviation (s.d.), standard deviation independent of the mean (SDIM), coefficient of variation (CV), successive variation (SV), average real variability (ARV), range, maximum, peak size and trough size of systolic BP in the Trial of Preventing Hypertension placebo arm (n=288). The average age of participants was 48 years. Mean systolic BP was 133.5 mm Hg. VVV metrics were all significantly correlated (P<0.001). Correlations between s.d., SDIM, CV and range and between ARV and SV were ≥0.90. Kappa statistics between quintiles of SD, SDIM, CV and range and between ARV and SV were ≥0.80. In studies of the relationship of VVV with health outcomes, we recommend reporting results for one of the metrics of overall variability (s.d., SDIM, CV), one of the metrics of variability between consecutive visits (SV, ARV), and one or more of the metrics of extreme values at a single visit (maximum, peak size, trough size).

The effect of rest days on injury rates.

Despite the importance of recuperation, few have studied the impact of rest periods on injury prevention. We determined the effect of rest days (breaks) on injury rates and treatments using electronic injury records from an acrobatic circus company that employs former world-class athletes as acrobats. To account for accumulated fatigue, we considered breaks across SD3 (third consecutive week of 1-day rest) to SD6 as a single exposure level (SD3-6), and vacation and DD (2-day rest) as a single exposure level. Medical attention injury rates were increased post- vs pre-break {rate ratio 1.45 [95% confidence intervals (95% CI): 1.22-1.73]} with less of an effect for 1-day time loss [1.25 (95% CI: 0.58-2.67)] and 15-day time loss [1.10 (95% CI: 0.26-4.56)]. However, the increase in injury rate post break for SD3-6 was similar to that of DD-Vacation (P=0.48, 0.53, and 0.65) for medical attention, and both ≥1 day and ≥15 days time loss, respectively. The increase in the number of treatments post-break was less for SD3-6 vs DD-vacation. Our findings suggest that 2-day breaks every four to 6 weeks may be sufficient to avoid an increasing injury rate due to cumulative fatigue in professional acrobatic circus artists.

Fatty fish, marine omega-3 fatty acids and incidence of heart failure.

BACKGROUND/OBJECTIVES: Marine omega-3 fatty acids have beneficial effects on cardiovascular risk factors. Consumption of fatty fish and marine omega-3 has been associated with lower rates of cardiovascular diseases. We examined the association of fatty fish and marine omega-3 with heart failure (HF) in a population of middle-aged and older women. SUBJECTS/METHODS: Participants in the Swedish Mammography Cohort aged 48-83 years completed 96-item food-frequency questionnaires. Women without any history of HF, myocardial infarction or diabetes at baseline (n=36,234) were followed from 1 January 1998 until 31 December 2006 for HF hospitalization or mortality through Swedish inpatient and cause-of-death registers; 651 women experienced HF events. Cox proportional hazards models accounting for age and other confounders were used to calculate incidence rate ratios (RR) and 95% confidence intervals (CI). RESULTS: Compared with women who did not eat fatty fish, RR were 0.86 (95% CI: 0.67, 1.10) for <1 serving per week, 0.80 (95% CI: 0.63, 1.01) for 1 serving per week, 0.70 (95% CI: 0.53, 0.94) for 2 servings per week and 0.91 (95% CI: 0.59, 1.40) for >or=3 servings per week (P(trend)=0.049). RR across quintiles of marine omega-3 fatty acids were 1 (reference), 0.85 (95% CI: 0.67, 1.07), 0.79 (95% CI: 0.61, 1.02), 0.83 (95% CI 0.65, 1.06) and 0.75 (95% CI: 0.58, 0.96) (P(trend)=0.04). CONCLUSION: Moderate consumption of fatty fish (1-2 servings per week) and marine omega-3 fatty acids were associated with a lower rate of first HF hospitalization or death in this population.

Dietary glycemic index, dietary glycemic load and mortality among men with established cardiovascular disease.

BACKGROUND/OBJECTIVES: In men with established cardiovascular disease, the effect of diets with high glycemic index (GI) and glycemic load (GL) is unknown. We tested the hypothesis that diets with higher GI and GL are associated with increased mortality in men with established cardiovascular disease. SUBJECTS/METHODS: We measured dietary GI and GL using food-frequency questionnaires in 4617 men, 45-79 years old, with a history of cardiovascular disease. The men were followed for cardiovascular mortality (6-year follow-up, 608 cases) and all-cause mortality (8-year follow-up, 1303 cases) using the Swedish cause-of-death and death registers. We used Cox models with age as the timescale and adjusted for body mass index, physical activity, history of hypertension and diabetes, family history of myocardial infarction, aspirin use, cigarette smoking and dietary factors to estimate incidence rate ratios (RRs). RESULTS: Comparing top to bottom quartiles of dietary GI, the RR for cardiovascular mortality was 0.86 (95% confidence interval (CI) 0.67-1.10, P for linear trend=0.21), and the RR for all-cause mortality was 1.00 (95% CI 0.85-1.19, P for linear trend=0.87). Compared to quartile 1, the RR for men with dietary GL in quartile 4 was 1.02 (95% CI 0.70-1.49, P for linear trend=0.81) for cardiovascular and 1.15 (95% CI 0.89-1.49, P for linear trend=0.20) for all-cause mortality. CONCLUSIONS: In this population of men with prior cardiovascular disease, dietary GI and GL were not associated with cardiovascular or all-cause mortality.

Maternal smoking during pregnancy and child overweight: systematic review and meta-analysis.

OBJECTIVE: Perform a systematic review of studies reporting on the association between maternal prenatal cigarette smoking and child overweight. DESIGN: Meta-analysis of observational studies. DATA SOURCES: Medline search and review of reference lists among studies published through June 2006. REVIEW METHODS: Included studies reported an association between maternal smoking during pregnancy and risk of overweight among children at least 2 years of age. We did not include in the meta-analysis studies that provided only a continuous measure of adiposity, although those studies are discussed separately. RESULTS: Based on results of 84 563 children reported in 14 observational studies, children whose mothers smoked during pregnancy were at elevated risk for overweight (pooled adjusted odds ratio (OR) 1.50, 95% CI: 1.36, 1.65) at ages 3-33 years, compared with children whose mothers did not smoke during pregnancy. The pooled estimate from unadjusted odds ratios (OR 1.52, 95% CI: 1.36, 1.69) was similar to the adjusted estimate, suggesting that sociodemographic and behavioral differences between smokers and nonsmokers did not explain the observed association. Although we observed evidence for publication bias, simulating a symmetric set of studies yielded a similar estimate (OR 1.40, 95% CI: 1.26, 1.55). CONCLUSIONS: Prenatal smoking exposure appears to increase rates of overweight in childhood. In parts of the world undergoing the epidemiologic transition, the continuing increase in smoking among young women could contribute to spiraling increases in rates of obesity-related health outcomes in the 21st century.

HbA1c measured in stored erythrocytes and mortality rate among middle-aged and older women.

AIMS/HYPOTHESIS: Diabetes is known to increase mortality rate, but the degree to which mild hyperglycaemia may be associated with the risk of death is uncertain. We examined the association between HbA1c measured in stored erythrocytes and mortality rate in women with and without diabetes. METHODS: We conducted a cohort study of 27,210 women>or=45 years old with no history of cardiovascular disease or cancer who participated in the Women's Health Study, a randomised trial of vitamin E and aspirin. RESULTS: Over a median of 10 years of follow-up, 706 women died. Proportional hazards models adjusted for age, smoking, hypertension, blood lipids, exercise, postmenopausal hormone use, multivitamin use and C-reactive protein were used to estimate the relative risk of mortality. Among women without a diagnosis of diabetes and HbA1c<5.60%, those in the top quintile (HbA1c 5.19-5.59%) had a relative risk of mortality of 1.28 (95% CI 0.98-1.69, p value for linear trend=0.14) compared with those with HbA1c 2.27-4.79%. Women with HbA1c 5.60-5.99% and no diagnosis of diabetes had a 54% increased risk of mortality (95% CI 1-136%) compared with those with HbA1c 2.27-4.79%. HbA1c was significantly associated with mortality across the range 4.50-7.00% (p value for linear trend=0.02); a test of deviation from linearity was not statistically significant (p=0.67). Diabetic women had more than twice the mortality risk of non-diabetic women. CONCLUSIONS/INTERPRETATION: This study provides further evidence that chronic mild hyperglycaemia, even in the absence of diagnosed diabetes, is associated with increased risk of mortality. ClinicalTrials.gov ID no.: NCT00000479.

A vital role for voltage-dependent potassium channels in dopamine transporter-mediated 6-hydroxydopamine neurotoxicity.

6-Hydroxydopamine (6-OHDA), a neurotoxic substrate of the dopamine transporter (DAT), is widely used in Parkinson's disease models. However, the molecular mechanisms underlying 6-OHDA's selectivity for dopamine neurons and the injurious sequelae that it triggers are not well understood. We tested whether ectopic expression of DAT induces sensitivity to 6-OHDA in non-dopaminergic rat cortical neurons and evaluated the contribution of voltage-dependent potassium channel (Kv)-dependent apoptosis to the toxicity of this compound in rat cortical and midbrain dopamine neurons. Cortical neurons expressing DAT accumulated dopamine and were highly vulnerable to 6-OHDA. Pharmacological inhibition of DAT completely blocked this toxicity. We also observed a p38-dependent Kv current surge in DAT-expressing cortical neurons exposed to 6-OHDA, and p38 antagonists and Kv channel blockers were neuroprotective in this model. Thus, DAT-mediated uptake of 6-OHDA recruited the oxidant-induced Kv channel dependent cell death pathway present in cortical neurons. Finally, we report that 6-OHDA also increased Kv currents in cultured midbrain dopamine neurons and this toxicity was blocked with Kv channel antagonists. We conclude that native DAT expression accounts for the dopamine neuron specific toxicity of 6-OHDA. Following uptake, 6-OHDA triggers the oxidant-associated Kv channel-dependent cell death pathway that is conserved in non-dopaminergic cortical neurons and midbrain dopamine neurons.

Effects of oral magnesium supplementation on glycaemic control in Type 2 diabetes: a meta-analysis of randomized double-blind controlled trials.

AIMS: The aim of this study was to assess the evidence on the effect of oral magnesium supplementation on glycaemic control in patients with Type 2 diabetes. METHODS: We searched the electronic databases of medline, embase and the Cochrane Controlled Trials Register up to January 2005. We identified nine randomized double-blind controlled trials with a total of 370 patients with Type 2 diabetes and of duration 4-16 weeks. The median dose of oral magnesium supplementation was 15 mmol/day (360 mg/day) in the treatment groups. The primary outcome was glycaemic control, as measured by glycated haemoglobin (HbA(1c)) or fasting blood glucose levels; the secondary outcomes included body mass index, blood pressure (BP) and lipids. Using a random-effects model, we calculated the weighted mean differences (WMD) and 95% confidence interval (CI). RESULTS: After a median duration of 12 weeks, the weighted mean post-intervention fasting glucose was significantly lower in the treatment groups compared with the placebo groups [-0.56 mmol/l (95% CI, -1.10 to -0.01); P for heterogeneity = 0.02]. The difference in post-intervention HbA(1c) between magnesium supplementation groups and control groups was not significant [-0.31% (95% CI, -0.81 to 0.19); P for heterogeneity = 0.10]. Neither systolic nor diastolic BP was significantly changed. Magnesium supplementation increased on high-density lipoprotein (HDL) cholesterol levels [0.08 mmol/l (95% CI, 0.03 to 0.14); P for heterogeneity = 0.36] but had no effect on total cholesterol, low-density lipoprotein (LDL) cholesterol and triglyceride. CONCLUSIONS: Oral magnesium supplementation for 4-16 weeks may be effective in reducing plasma fasting glucose levels and raising HDL cholesterol in patients with Type 2 diabetes, although the long-term benefits and safety of magnesium treatment on glycaemic control remain to be determined.

Apoptotic surface delivery of K+ channels.

Apoptosis in cortical neurons requires efflux of cytoplasmic potassium mediated by a surge in Kv2.1 channel activity. Pharmacological blockade or molecular disruption of these channels in neurons prevents apoptotic cell death, while ectopic expression of Kv2.1 channels promotes apoptosis in non-neuronal cells. Here, we use a cysteine-containing mutant of Kv2.1 and a thiol-reactive covalent inhibitor to demonstrate that the increase in K+ current during apoptosis is due to de novo insertion of functional channels into the plasma membrane. Biotinylation experiments confirmed the delivery of additional Kv2.1 protein to the cell surface following an apoptotic stimulus. Finally, expression of botulinum neurotoxins that cleave syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) blocked upregulation of surface Kv2.1 channels in cortical neurons, suggesting that target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins support proapoptotic delivery of K+ channels. These data indicate that trafficking of Kv2.1 channels to the plasma membrane causes the apoptotic surge in K+ current.

Nitric oxide-induced changes in intracellular zinc homeostasis are mediated by metallothionein/thionein.

We hypothesized that metallothionein (MT), a cysteine-rich protein with a strong affinity for Zn(2+), plays a role in nitric oxide (NO) signaling events via sequestration or release of Zn(2+) by the unique thiolate clusters of the protein. Exposing mouse lung fibroblasts (MLF) to the NO donor S-nitrosocysteine resulted in 20-30% increases in fluorescence of the Zn(2+)-specific fluorophore Zinquin that were rapidly reversed by the Zn(2+) chelator N,N,N',N'-tetrakis-(2-pyridylmethyl)ethylenediamine. The absence of a NO-mediated increase in labile Zn(2+) in MLF from MT knockouts and its restoration after MT complementation by adenoviral gene transfer inferred a critical role for MT in the regulation of Zn(2+) homeostasis by NO. Additional data obtained in sheep pulmonary artery endothelial cells suggested a role for the apo form of MT, thionein (T), as a Zn(2+)-binding protein in intact cells, as overexpression of MT caused inhibition of NO-induced changes in labile Zn(2+) that were reversed by Zn(2+) supplementation. Furthermore, fluorescence-resonance energy-transfer data showed that overexpression of green fluorescent protein-modified MT prevented NO-induced conformational changes, which are indicative of Zn(2+) release from thiolate clusters. This effect was restored by Zn(2+) supplementation. Collectively, these data show that MT mediates NO-induced changes in intracellular Zn(2+) and suggest that the ratio of MT to T can regulate Zn(2+) homeostasis in response to nitrosative stress.

Visualization of neuropeptide expression, transport, and exocytosis in Drosophila melanogaster.

Neuropeptides affect an extremely diverse set of physiological processes. Neuropeptides are often coreleased with neurotransmitters but, unlike neurotransmitters, the neuropeptide target cells may be distant from the site(s) of secretion. Thus, it is often difficult to measure the amount of neuropeptide release in vivo by electrophysiological methods. Here we establish an in vivo system for studying the developmental expression, processing, transport, and release of neuropeptides. A GFP-tagged atrial natriuretic factor fusion (preproANF-EMD) was expressed in the Drosophila nervous system with the panneural promoter, elav. During embryonic development, proANF-EMD was first seen to accumulate in synaptic regions of the CNS in stage 17 embryos. By the third instar larval stage, highly fluorescent neurons were evident throughout the CNS. In the adult, fluorescence was pronounced in the mushroom bodies, antennal lobe, and the central complex. At the larval neuromuscular junction, proANF-EMD was concentrated in nerve terminals. We compared the release of proANF-EMD from synaptic boutons of NMJ 6/7, which contain almost exclusively glutamate-containing clear vesicles, to those of NMJ 12, which include the peptidergic type III boutons. Upon depolarization, approximately 60% of the tagged neuropeptide was released from NMJs of both muscles in 15 min, as assayed by decreased fluorescence. Although the elav promoter was equally active in the motor neurons that innervate both NMJs 6/7 and 12, NMJ 12 contained 46-fold more neuropeptide and released much more proANF-EMD during stimulation than did NMJ 6/7. Our results suggest that peptidergic neurons have an enhanced ability to accumulate and/or release neuropeptides as compared to neurons that primarily release classical neurotransmitters.

Acid prohormone sequence determines size, shape, and docking of secretory vesicles in atrial myocytes.

How vesicles are born in the trans-Golgi network and reach their docking sites at the plasma membrane is still largely unknown and is investigated in the present study on live, primary cultured atrial cardiomyocytes. Secretory vesicles (n=422) are visualized by expressing fusion proteins of proatrial natriuretic peptide (proANP) and green fluorescent protein. Myocytes expressing fusion proteins with intact proANP display two populations of fluorescent vesicles with apparent diameters of 120 and 175 nm, moving at a top velocity of 0.3 microm/s. The number of docked vesicles is significantly correlated with the number of mobile vesicles (r=0.71, P<0.0005). The deletion of the acidic N-terminal proANP[1-44] or point mutations (glu(23,24)-->gln(23,24)) change size and shape-but not velocity-of the vesicles, and, strikingly, abolish their docking at the plasma membrane. The shapes thus change from spheres to larger, irregular floppy bags or vesicle trains. Deletion of the C-terminal proANP[45-127], where the ANP and its disulfide bond reside, does not change size, shape, docking, or velocity of the mobile vesicles. The N-terminal acid calcium-binding sequence of proANP is known to cause protein aggregation at the high calcium concentration prevailing in the trans-Golgi network. Therefore, these results indicate that amino acid residues favoring cargo aggregation are critically important in shaping the secretory vesicles and determining their fate-docking or not docking-at the plasma membrane. The full text of this article is available at http://www.circresaha.org.

Dynamic in vivo interactions among Myc network members.

Members of the Myc oncoprotein network (c-Myc, Max, and Mad) play important roles in proliferation, differentiation, and apoptosis. We expressed chimeric green fluorescent protein (GFP) fusions of c-Myc, Max, and three Mad proteins in fibroblasts. Individually, c-Myc and Mad proteins localized in subnuclear speckles, whereas Max assumed a homogeneous nuclear pattern. These distributions were co-dominant and dynamic, however, as each protein assumed the pattern of its heterodimeric partner when the latter was co-expressed at a higher level. Deletion mapping of two Mad members, Mad1 and Mxi1, demonstrated that the domains responsible for nuclear localization and speckling are separable. A non-speckling Mxi1 mutant was also less effective as a transcriptional repressor than wild-type Mxi1. c-Myc nuclear speckles were distinct from SC-35 domains involved in mRNA processing. However, in the presence of co-expressed Max, c-Myc, but not Mad, co-localized to a subset of SC-35 loci. These results show that Myc network proteins comprise dynamic subnuclear structures and behave co-dominantly when co-expressed with their normal heterodimerization partners. In addition, c-Myc-Max heterodimers, but not Max-Mad heterodimers, localize to foci actively engaged in pre-mRNA transcription/processing. These findings suggest novel means by which Myc network members promote transcriptional activation or repression.

Agonist-dependent traffic of raft-associated Ras and Raf-1 is required for activation of the mitogen-activated protein kinase cascade.

Stimulation of HIRcB fibroblasts with insulin leads to accumulation of active components of the mitogen-activated protein kinase cascade in endocytic compartments. However, the factors that regulate the mobilization of these components through the endocytic pathway and the relevance of this event to cellular signaling remain unclear. Here we report that Ras proteins are associated with lipid rafts in resting HIRcB fibroblasts. Ras is rapidly internalized into the endocytic compartment following stimulation with insulin. The redistribution of Ras is independent of its activation. Attachment of the C-terminal 20 amino acids of Ha-Ras to green fluorescent protein was sufficient to target this construct to the same loci as the endogenous Ras protein, indicating that Ras distribution is a consequence of the association of its lipid modified C terminus with membranes. Depletion of plasma membrane cholesterol delocalized Ras and blocked insulin-dependent Ras traffic. Cholesterol depletion also blocked insulin-dependent phosphorylation of MEK and mitogen-activated protein kinase (MAPK) but had no effects on the translocation and activation of Raf-1. A second inhibitor of endocytosis, cytochalasin D, also blocked insulin-dependent MAPK phosphorylation. Taken together, these results suggest that mobilization of active Raf-1 through the endocytic compartment is required for completion of the MAPK cascade.

Free intracellular Mg(2+) concentration and inhibition of NMDA responses in cultured rat neurons.

1. Intracellular Mg(2+) (Mg(2+)(i)) blocks single-channel currents and modulates the gating kinetics of NMDA receptors. However, previous data suggested that Mg(2+)(i) inhibits whole-cell current less effectively than predicted from excised-patch measurements. We examined the basis of this discrepancy by testing three hypothetical explanations. 2. To test the first hypothesis, that control of free Mg(2+)(i) concentration ([Mg(2+)](i)) during whole-cell recording was inadequate, we measured [Mg(2+)](i) using mag-indo-1 microfluorometry. The [Mg(2+)](i) measured in cultured neurons during whole-cell recording was similar to the pipette [Mg(2+)] measured in vitro, suggesting that [Mg(2+)](i) was adequately controlled. 3. To test the second hypothesis, that open-channel block by Mg(2+)(i) was modified by patch excision, we characterised the effects of Mg(2+)(i) using cell-attached recordings. We found the affinity and voltage dependence of open-channel block by Mg(2+)(i) similar in cell-attached and outside-out patches. Thus, the difference between Mg(2+)(i) inhibition of whole-cell and of patch currents cannot be attributed to a difference in Mg(2+)(i) block of single-channel current. 4. The third hypothesis tested was that the effect of Mg(2+)(i) on channel gating was modified by patch excision. Results of cell-attached recording and modelling of whole-cell data suggest that the Mg(2+)(i)-induced stabilisation of the channel open state is four times weaker after patch excision than in intact cells. This differential effect of Mg(2+)(i) on channel gating explains why Mg(2+)(i) inhibits whole-cell NMDA responses less effectively than patch responses.

Restriction of secretory granule motion near the plasma membrane of chromaffin cells.

We used total internal reflection fluorescence microscopy to study quantitatively the motion and distribution of secretory granules near the plasma membrane (PM) of living bovine chromaffin cells. Within the approximately 300-nm region measurably illuminated by the evanescent field resulting from total internal reflection, granules are preferentially concentrated close to the PM. Granule motion normal to the substrate (the z direction) is much slower than would be expected from free Brownian motion, is strongly restricted over tens of nanometer distances, and tends to reverse directions within 0.5 s. The z-direction diffusion coefficients of granules decrease continuously by two orders of magnitude within less than a granule diameter of the PM as granules approach the PM. These analyses suggest that a system of tethers or a heterogeneous matrix severely limits granule motion in the immediate vicinity of the PM. Transient expression of the light chains of tetanus toxin and botulinum toxin A did not disrupt the restricted motion of granules near the PM, indicating that SNARE proteins SNAP-25 and VAMP are not necessary for the decreased mobility. However, the lack of functional SNAREs on the plasma or granule membranes in such cells reduces the time that some granules spend immediately adjacent to the PM.

Independent regulation of cardiac Kv4.3 potassium channel expression by angiotensin II and phenylephrine.

Hypertrophied cardiac myocytes exhibit prolonged action potentials and decreased transient outward potassium current (I(to)). Because Kv4.3 is a major contributor to I:(to), we studied regulation of its expression in neonatal rat cardiac myocytes in response to the known stimulators of cardiac myocyte hypertrophy, angiotensin II (Ang II) and phenylephrine (PE). RNase protection assays and immunoblots revealed that Ang II and PE each downregulate Kv4.3 mRNA and protein. However, although PE induces a faster and more extensive hypertrophic response than Ang II, the PE effect on Kv4.3 mRNA develops slowly and is sustained, whereas Ang II rapidly and transiently decreases Kv4.3 mRNA expression. Turnover measurements revealed that Kv4.3 mRNA is very stable, with a half-life >20 hours. This suggests that Ang II must destabilize the channel mRNA. In contrast, PE does not affect the rate of Kv4.3 mRNA degradation. To test for transcriptional regulation, the 5' flanking region of the rat Kv4.3 gene was cloned, and Kv4.3 promoter-reporter constructs were expressed in cardiac myocytes. Whereas Ang II was found to have no effect on transcription, PE inhibits Kv4.3 promoter activity. Pharmacological experiments also indicate that PE and Ang II act independently to downregulate Kv4.3 gene expression. Thus, regulation of Kv4.3 gene expression is not a simple secondary response to hypertrophy. Rather, Ang II and PE use different mechanisms to decrease Kv4.3 channel expression in neonatal rat cardiac myocytes.

Kvbeta subunits increase expression of Kv4.3 channels by interacting with their C termini.

Auxiliary Kvbeta subunits form complexes with Kv1 family voltage-gated K(+) channels by binding to a part of the N terminus of channel polypeptide. This association influences expression and gating of these channels. Here we show that Kv4.3 proteins are associated with Kvbeta2 subunits in the brain. Expression of Kvbeta1 or Kvbeta2 subunits does not affect Kv4.3 channel gating but increases current density and protein expression. The increase in Kv4.3 protein is larger at longer times after transfection, suggesting that Kvbeta-associated channel proteins are more stable than those without the auxiliary subunits. This association between Kv4.3 and Kvbeta subunits requires the C terminus but not the N terminus of the channel polypeptide. Thus, Kvbeta subunits utilize diverse molecular interactions to stimulate the expression of Kv channels from different families.

TRH regulates Kv1.5 gene expression through a Galphaq-mediated PLC-independent pathway.

Thyrotropin-releasing hormone (TRH) decreases transcription of the Kv1.5 K(+) channel gene in GH(3) pituitary cells. Here, we examine whether TRH utilizes Gq activated phospholipase C, Gs or Gi to produce this response. We report that expression of constitutively active Galphaq mimicked and occluded the TRH effect. In contrast, expression of activated Galpha(S) or Galpha(i2) had no effect on Kv1. 5 mRNA expression. Furthermore, pertussis and cholera toxins failed to block the TRH-induced decrease in channel mRNA. Surprisingly, despite the role of Gq, the phospholipase C inhibitor U73122 did not alter down-regulation of channel mRNA by TRH, although it abolished the TRH-induced increase in intracellular [Ca(2+)] and up-regulation of c-fos mRNA. Furthermore, depletion of an intracellular Ca(2+) pool or inhibition of protein kinase C did not block the TRH-induced decrease in Kv1.5 mRNA. These results indicate that TRH-induced down-regulation of Kv1.5 gene expression is mediated by Galphaq proteins, but does not require PLC activation.