Acceptability of human papillomavirus self testing in female adolescents.

OBJECTIVES: To develop scales assessing acceptability of human papillomavirus (HPV) testing in adolescents, to compare acceptability of self to clinician testing, and to identify adolescent characteristics associated with acceptability. METHODS: Female adolescents 14-21 years of age attending a hospital based teen health centre self collected vaginal samples and a clinician, using a speculum, collected cervicovaginal samples for HPV DNA. Acceptability of and preferences for self and clinician testing were assessed at baseline and 2 week visits. RESULTS: The mean age of the 121 participants was 17.8 years and 82% were black. The acceptability scales demonstrated good internal consistency, reliability, test-retest reliability, and factorial validity. Scores were significantly lower for self testing than clinician testing on the acceptability scale and three subscales measuring trust of the test result, confidence in one's ability to collect a specimen, and perceived effects of testing (p < 0.01). Of those who reported a preference, 73% preferred clinician to self testing. Acceptability scores for both self and clinician testing increased significantly pre-examination to post-examination (p < 0.01). Multivariable analyses demonstrated that race was independently associated with pre-examination and post-examination acceptability of self testing, and that sexual behaviours and gynaecological experiences were associated with specific acceptability subscales. CONCLUSIONS: This sample of adolescents found clinician testing for HPV to be more acceptable than self testing and preferred clinician to self testing. If self testing for HPV is offered in the future, clinicians should not assume that adolescent patients will prefer self testing. Instead, they should educate adolescents about available testing options and discuss any concerns regarding self collection technique or accuracy of test results.

beta-adrenergic stimulation restores the Ca transient of ventricular myocytes lacking t-tubules.

beta-adrenergic stimulation helps to synchronize Ca release in myocytes from failing hearts. Transverse (t-) tubules, which synchronize Ca release in normal cells and contain many of the elements of the beta-adrenergic pathway, may be depleted in such cells. The objective of the present study was to determine whether beta-adrenergic stimulation could reverse the desynchronization of Ca release observed in detubulated ventricular myocytes. The effect of isoprenaline (0.5 microM) on control and detubulated rat ventricular myocytes was investigated. Ca transients were monitored using whole-cell fluorescence and confocal microscopy, and Ca current recorded using the patch-clamp technique. Immunocytochemistry was used to investigate phospholamban (PLB) phosphorylation. Detubulation reduces and slows the Ca transient; these effects were reversed by isoprenaline. This restoration was associated with partial reversal of the desynchronization of Ca release that occurs in detubulated cells. Sarcoplasmic reticulum Ca load increased by the same amount in normal and detubulated cells, but Ca current increased less in detubulated cells (64%) than in control cells (124%) in response to isoprenaline. The pattern and extent of cAMP-dependent protein kinase and CaMKII-induced phosphorylation of PLB in response to isoprenaline was the same in both cell types. Thus, the beta-adrenergic pathway is functional in the absence of t-tubules; such stimulation appears to increase the speed of propagation of Ca via Ca-induced Ca release between adjacent clusters of ryanodine receptors, which may be relevant in pathological conditions, such as heart failure, in which t-tubules are depleted. The data also suggest that the Ca current responds to local signaling pathways, which are better coupled to the channel in the t-tubules than at the surface membrane, whereas PLB responds to whole-cell signaling.

Biphasic effects of hyposmotic challenge on excitation-contraction coupling in rat ventricular myocytes.

The effects of short (1 min) and long (7-10 min) exposure to hyposmotic solution on excitation-contraction coupling in rat ventricular myocytes were studied. After short exposure, the action potential duration at 90% repolarization (APD(90)), the intracellular Ca(2+) concentration ([Ca(2+)](i)) transient amplitude, and contraction increased, whereas the L-type Ca(2+) current (I(Ca, L)) amplitude decreased. Fractional sarcoplasmic reticulum (SR) Ca(2+) release increased but SR Ca(2+) load did not. After a long exposure, I(Ca,L), APD(90), [Ca(2+)](i) transient amplitude, and contraction decreased. The abbreviation of APD(90) was partially reversed by 50 microM DIDS, which is consistent with the participation of Cl(-) current activated by swelling. After 10-min exposure to hyposmotic solution in cells labeled with di-8-aminonaphthylethenylpyridinium, t-tubule patterning remained intact, suggesting the loss of de-t-tubulation was not responsible for the fall in I(Ca,L). After long exposure, Ca(2+) load of the SR was not increased, and swelling had no effect on the site-specific phosphorylation of phospholamban, but fractional SR Ca(2+) release was depressed. The initial positive inotropic response to hyposmotic challenge may be accounted for by enhanced coupling between Ca(2+) entry and release. The negative inotropic effect of prolonged exposure can be accounted for by shortening of the action potential duration and a fall in the I(Ca,L) amplitude.

Quantitative analysis of Na+-Ca2+ exchanger expression in guinea-pig heart.

In previous studies, regional variations in the expression of the Na+-Ca2+ exchanger (NCX) have been examined qualitatively in human heart using the C2C12 monoclonal antibody [Wang, J., Schwinger, R.H., Frank, K., Muller-Ehmsen, J., Martin-Vasallo, P., Pressley, T.A., Xiang, A., Erdmann, E. & McDonough, A.A. (1996) J. Clin. Invest. 98, 1650-1658]. Although NCX expression was found to be significantly lower in the atria compared to the septum, no significant differences were found between atrial and ventricular tissue. NCX has been located in the general sarcolemma and t-tubules of ventricular muscle and as t-tubules are sparse in atrial tissue compared to ventricular tissue, it is surprising that NCX expression was found to be similar in both atria and ventricles [Wang et al. (1996)]. To reinvestigate this, we have used SDS/PAGE and a quantitative Western blotting technique to determine the pattern of expression of NCX in guinea-pig heart in tissue samples from left atrium, right atrium, septum, left ventricle and right ventricle. NCX protein expression was 17.5 +/- 3.9 pmol.mg-1 of protein in the left atrium and 29.2 +/- 6.1 pmol.mg-1 of protein in the right atrium, which were both significantly lower (P < 0.05) than NCX expression in the septum, left ventricle and right ventricle (64.7 +/- 15.2, 76.8 +/- 19.5 and 69.4 +/- 14.1 pmol.mg-1 of protein, respectively, n = 7). These differences in NCX expression may reflect variations in the cellular location of NCX protein in these regions. To study this, we used confocal immunofluorescence of single isolated myocytes to examine differences in the proportion of fluorescent staining on the general surface membrane compared with the interior of the cell (which presumably reflects a t-tubular location). We found that the general membrane staining was 79.0 +/- 1.2% in cells from the atria which was significantly higher (P < 0. 001) than that seen in cells from the septum, left ventricle and right ventricle, with 48.1 +/- 1.1%, 48.2 +/- 1.8% and 45.6 +/- 1.3%, respectively (n = 20). These results illustrate a similar pattern of NCX expression in guinea-pig and human, with expression in atrial tissue significantly lower than in ventricular tissue. However, the cellular location of NCX differs regionally; in atrial tissue, the majority of the NCX protein is located in the general sarcolemma whereas in ventricular and septal tissue, approximately 50% of NCX protein is located within the cell (presumably at the level of the t-tubules).

Sites on the cytoplasmic region of phospholamban involved in interaction with the calcium-activated ATPase of the sarcoplasmic reticulum.

Proton NMR studies have shown that when a peptide corresponding to the N-terminal region of phospholamban, PLB(1-20), interacts with the Ca2+ATPase of the sarcoplasmic reticulum, SERCA1a, docking involves the whole length of the peptide. Phosphorylation of Ser16 reduced the affinity of the peptide for the pump by predominantly affecting the interaction with the C-terminal residues of PLB(1-20). In the phosphorylated peptide weakened interaction occurs with residues at the N-terminus of PLB(1-20). PLB(1-20) is shown to interact with a peptide corresponding to residues 378-405 located in the cytoplasmic region of SERCA2a and related isoforms. This interaction involves the C-terminal regions of both peptides and corresponds to that affected by phosphorylation. The data provide direct structural evidence for complex formation involving residues 1-20 of PLB. They also suggest that phospholamban residues 1-20 straddle separate segments of the cytoplasmic domain of SERCA with the N-terminus of PLB associated with a region other than that corresponding to SERCA2a(378-405).

Effects of the protein kinase A inhibitor H-89 on Ca2+ regulation in isolated ferret ventricular myocytes.

We investigated the effects of a protein kinase A (PKA) inhibitor, H-89 {N-[2-(p-bromocinnamylamino)ethyl]-5-iso-quinolinesulphonamide}, on Ca2+ regulation in Fura-2-loaded ferret myocytes. H-89 (10 micromol/l) decreased the amplitude of the Fura-2 transient to 28. 2+/-4.3% (P<0.001) of control and prolonged its duration, characterized by a decrease in the rate of decline of Ca2+ to diastolic levels: t1/2 increased from 311+/-35 ms to 547+/-43 ms (P<0.001, n=7). Reduced Ca2+ uptake by the sarcoplasmic reticulum (SR) in the presence of H-89 was also indicated by a decrease in the SR Ca2+ content, as assessed with caffeine. The apparent slowing of the SR Ca2+-ATPase was not caused by changes in phosphorylation of phospholamban (PLB). However, Ca2+ uptake in microsomal vesicles prepared from canine hearts and fast-twitch rat skeletal muscle (which lacks PLB) was decreased by 34.1 and 46.8% (n=3), respectively, suggesting that H-89 has a direct inhibitory effect on the SR Ca2+-ATPase. In electrophysiological experiments, 5.0 micromol/l H-89 decreased the L-type Ca2+ current (ICa) by 39.5% (n=6) and slowed the upstroke of the action potential and, in some cases, caused loss of excitability without changes in the resting membrane potential. In summary, data show that [Ca2+ ]i regulation, and hence contraction, is sustained by PKA-mediated phosphorylation, even in the absence of beta-agonists. However, the use of H-89 as a tool to study the role of this signalling pathway is limited by the non-specific effects of H-89 on the SR Ca2+-ATPase.

Cyclic AMP but not phosphorylation of phospholamban contributes to the slow inotropic response to stretch in ferret papillary muscle.

cAMP has been suggested to mediate the increased intracellular Ca2+ transient and contraction seen during the slow response to stretch in cardiac muscle. We measured cAMP in ferret papillary muscles stretched from 80-85% to 98% of their length at which maximum active tension is produced (Lmax) for 15 min. cAMP was significantly (P<0. 05) increased by 53% in muscles at the longer length which showed the slow response compared with controls. By contrast, in a population of muscles that were stretched but did not show the slow response, cAMP was not significantly different from that in muscles at the short length. Although cAMP can increase sarcoplasmic reticulum (SR) Ca2+ uptake by phosphorylation of phospholamban, we found no significant effect of stretch on phosphorylation of phospholamban at either Ser16 or Thr17. Further support for the hypothesis that cAMP is a mediator of the slow response was obtained by exposure of some muscles to the cell-permeable cAMP antagonist 8-bromo, adenosine 3',5'-cyclic monophosphorothioate, Rp isomer (Rp-8-Br-cAMPS, (2.5-10 microM). The slow response was reduced by 30% (P<0.05) in the presence of this antagonist. Our results not only provide evidence for the mediation of the slow response to stretch by cAMP, they also suggest that cAMP may rise in an intracellular compartment inaccessible to the SR.

Co-ordinated changes in cAMP, phosphorylated phospholamban, Ca2+ and contraction following beta-adrenergic stimulation of rat heart.

Concentration-dependent changes in cyclic AMP (cAMP), site-specific phosphorylation of phospholamban, the intracellular calcium ([Ca2+]i) transient and contraction were measured in isolated rat ventricular myocytes exposed to the beta-adrenoceptor agonist isoprenaline. Cyclic AMP was measured by [125I]-cAMP scintillation proximity assay, phosphorylation of phospholamban at Ser16 and Thr17 was assessed using a pair of site-specific polyclonal antibodies, and [Ca2+]i was monitored with the fluorescent dye fura 2. Cyclic AMP rose to twice basal levels in the presence of 10(-6) M isoprenaline. The maximum increase in phosphorylation at Ser16 and Thr17 of phospholamban was seen at 10(-7) M isoprenaline. At this stage Ser16 phosphorylation was six times higher, and Thr17 phosphorylation was three times higher than that recorded in the absence of isoprenaline. Phosphorylation at Ser16 correlated more closely with changes in the [Ca2+]i transient and contraction than did phosphorylation at Thr17. This is the first study of its kind to measure simultaneous changes in cAMP, the phosphorylation of phospholamban, the [Ca2+]i transient and contraction over a range of concentrations of beta-agonist. The results suggest that phosphorylation of phospholamban at Thr17 is of lesser physiological relevance to the effects of beta-adrenergic stimulation on the heart than phosphorylation at Ser16.

Preservation of the in vivo phosphorylation status of phospholamban in the heart: evidence for a site-specific difference in the dephosphorylation of phospholamban.

The phosphorylation status of the cardiac sarcoplasmic reticular (SR) protein phospholamban determines the activity of the SR Ca(2+)-ATPase. In order to predict SR Ca2+ transport in vivo, it is vital that techniques used to measure the phosphorylation status of phospholamban adequately clamp the endogenous kinases and phosphatases which modify phosphorylation during sample preparation. A recent study (Boateng, S., Seymour, A-M., Dunn, M., Yacoub, M., and Boheler, K. (1997) Biochem. Biophys. Res. Comm. 239, 701-705) has suggested that phosphatase inhibitors must be present in quenching media to prevent almost total dephosphorylation of phospholamban. We addressed this issue by assessing the effect of both kinase and phosphatase inhibition on site-specific phosphorylation of phospholamban in ferret ventricular muscle and isolated rat ventricular myocytes quenched with Laemmli sample buffer. Under these clearly defined quenching conditions in isolated myocytes, we demonstrated that the phosphorylation status of phospholamban was low under basal conditions, and high following exposure to the beta-agonist isoprenaline. The only significant effect of inhibitor inclusion in the quench solution was in isolated myocyte preparations where phosphatase inhibition increased phosphorylation at Ser16 by about a third. The differential effect of phosphatase inclusion on phosphorylation at Ser16 and Thr17 may indicate that different enzymes are involved in dephosphorylation of the two sites.

Depletion of Ca2+ from the sarcoplasmic reticulum of cardiac muscle prompts phosphorylation of phospholamban to stimulate store refilling.

Nonmuscle cells have almost ubiquitously evolved a mechanism to detect and prevent Ca2+ store depletion-store operated calcium entry. No such mechanism has, as yet, been reported in cardiac myocytes. However, it is conceivable that such a mechanism may play an important role in cardiac Ca2+ homeostasis to ensure the availability of sufficient stored Ca2+ to maintain normal excitation contraction coupling. We present data that confirms the presence of a mechanism that is able to monitor the Ca2+ load of the SR and initiate a signaling process to accelerate Ca2+ uptake by the SR when store depletion is detected. Depletion of SR Ca2+ activates a protein kinase, the principal SR substrate of which is phospholamban. Phosphorylation of this SR protein promotes Ca2+ pump activity and therefore store refilling. Furthermore, a protein kinase activity associated with the SR that is inhibited by Ca2+ ions has been identified. We have measured lumenal [Ca2+] by using a fluorescent Ca2+ indicator and found that by initiating Ca2+ uptake and increasing Ca2+ load, we can inhibit the protein kinase activity associated with the SR. This confirms that a protein kinase, that is regulated by lumenal [Ca2+], has been identified and represents part of a previously unidentified signalling cascade. This local feedback mechanism would allow the myocyte to detect and prevent SR Ca2+ load depletion.

Phosphorylation states of phospholamban.

Phospholamban is a small integral membrane protein of cardiac, smooth, and slow-twitch skeletal muscle sarcoplasmic reticulum that interacts with the Ca2+ pump of these organelles and inhibits Ca(2+)-pump activity while in the dephosphorylated form. Three sites of Ser/Thr phosphorylation have been identified in the primary sequence of phospholamban, at Ser-10, Ser-16, and Thr-17. In vitro studies indicate that these residues are phosphorylated by PKC (Ser-10), PKA, PKG or PKC (Ser-16), and CaM kinase II (Thr-17). Phosphorylation of Ser-16 (or Thr-17) is accompanied by an increase in Ca2+ pump activity in direct proportion to the stoichiometry of phosphorylation. Dual phosphorylation of both Ser-16 and Thr-17 does not cause any further stimulation of pump function over that achieved by stoichiometric phosphorylation of a single site. Examination of the pattern of phosphorylation in vivo has been aided by the generation of polyclonal antibodies specific for the phosphorylated forms of phospholamban. beta-Adrenergic stimulation of cardiac muscle results in phosphorylation of both Ser-16 and Thr-17. The time course of Ser-16 phosphorylation precedes Thr-17. The spatial distribution of Ser-16 and Thr-17 phosphorylated forms of phospholamban is not identical; phospholamban located in the nuclear membrane of a cardiac myocyte is phosphorylated exclusively on Ser-16, whereas phospholamban molecules in the SR membrane of the same cell are phosphorylated on Ser-16 and/or Thr-17. Finally, we have identified a novel stimulus for the phosphorylation of phospholamban. Ca2+ store depletion, achieved by exposure of myocytes to SERCA inhibitors, prompts the phosphorylation of phospholamban on Ser-16. This would be expected to increase Ca2+ uptake by the SR in an attempt to achieve the refilling of the SR.

Acidosis alters the phosphorylation of Ser16 and Thr17 of phospholamban in rat cardiac muscle.

The effect of acidosis on the phosphorylation of Ser16 and Thr17 of phospholamban in rat cardiac muscle has been investigated using phosphorylation-site-specific antibodies to this protein. Ventricular myocytes were stimulated at 0.5 Hz for 5 min, in either control (pH 7.4) or acid (pH 6.5) physiological salt solution, in the absence or presence of isoprenaline. Site-specific phosphorylation of phospholamban was determined by Western blotting. Acidosis reduced phosphorylation of Ser16 in the absence of isoprenaline, but did not alter the isoprenaline-induced phosphorylation of Ser16. In contrast, acidosis increased Thr17 phosphorylation in the absence and presence of isoprenaline. Buffering intracellular Ca2+ ([Ca2+]i) with BAPTA inhibited the increase in Thr17 phosphorylation during acidosis but had no effect on Ser16 phosphorylation. We conclude that acidosis can alter the phosphorylation of Ser16 and Thr17 by inhibition of protein kinase A, and by an acidosis-induced increase in [Ca2+]i and the subsequent activation of a Ca2+/calmodulin-dependent protein kinase, respectively. The possible effect of these changes in phosphorylation on the activity of the Ca2+-ATPase of the cardiac sarcoplasmic reticulum during acidosis is discussed.

Rate-dependent abbreviation of Ca2+ transient in rat heart is independent of phospholamban phosphorylation.

The mechanisms underlying the accelerated decline of the intracellular Ca2+ transient that occurs in cardiac muscle when stimulation rate is increased have been investigated in ventricular myocytes from rat hearts. Increasing stimulation rate from 0.1 to 0.5 and 1 Hz decreased the time taken for the Ca2+ transient to decline from its peak to 50% of its peak value in cells generating action potentials, when the duration of depolarization was held constant by voltage clamp, and when Na/Ca exchange was inhibited. The sarcoplasmic reticulum Ca2+ adenosinetriphosphatase inhibitor thapsigargin inhibited rate-dependent abbreviation of the Ca2+ transient. However, neither a chemical inhibitor of Ca(2+)-calmodulin-dependent protein kinase II (KN62) nor a peptide inhibitor of this enzyme (calmodulin-binding domain peptide) had a significant effect on rate-dependent abbreviation of the Ca2+ transient. Analysis of the phosphorylation of the regulatory sites Ser16 and Thr17 of phospholamban showed no significant change in phosphorylation with changes of stimulation rate. These data suggest that rate-dependent shortening of the Ca2+ transient is due predominantly to enhanced Ca2+ uptake by the sarcoplasmic reticulum without changes in phospholamban phosphorylation.

Translation of Ser16 and Thr17 phosphorylation of phospholamban into Ca 2+-pump stimulation.

Stimulation of cardiac sarcoplasmic reticulum Ca 2+-pump activity is achieved by phosphorylation of the oligomeric protein phospholamban at either Ser16 or Thr17. The altered mobility of phosphorylated forms of pentameric phospholamban has been utilized to demonstrate that the mechanisms of phosphorylation of the two sites differ. Phosphorylation of Ser16 by the AMP-dependent protein kinase proceeds via a random mechanism [Li, Wang and Colyer (1990) Biochemistry 29, 4535-4540], whereas phosphorylation of Thr17 by calmodulin-dependent protein kinase is shown here to proceed via a co-operative mechanism. This co-operative reaction mechanism was unaffected by the phosphorylation status of Ser16. These two mechanisms of phosphorylation generate very different phosphoprotein profiles depending on whether the Ser16 or Thr17 residue is phosphorylated. The translation of these patterns of phosphorylation into Ca 2+-pump function was reviewed using a fluorimetric Ca 2+-indicator dye, fluo-3, to measure Ca2+ uptake by cardiac sarcoplasmic reticulum vesicles. The rate of Ca2+ accumulation, which parallels Ca 2+-pump activity, was stimulated in proportion with the stoichiometry of phospholamban phosphorylation, irrespective of whether phosphorylation was on Ser16 or Thr17.

Conformational effects of serine phosphorylation in phospholamban peptides.

We have employed one- and two-dimensional 1H-NMR spectroscopy to study the effects of serine phosphorylation on peptide conformations, using cardiac phospholamban as a model system. The non-phosphorylated phospholamban 1-20 peptide has few restraints on the conformations available to it in aqueous solution. Phosphorylation at Ser16 results in greater constraints being placed on the region encompassing Arg14-Thr17, particularly at neutral pH when the phosphate group is in the di-anionic form. These conformational restrictions arise from specific interactions between the side-chain of Arg14 and the phosphate group. While substitution of phosphothreonine at position 16 causes generally similar effects to phosphoserine, aspartic acid has little effect. The results are compared with phosphorylation effects in other systems, including cardiac troponin I.