New benzodiazepine and Z-hypnotic users and disability pension: an eight-year nationwide observational follow-up study.

OBJECTIVE: To compare how newly initiated treatment with benzodiazepines, Z-hypnotics or both associates with the reception of disability pension among 40,661 individuals of a working age. DESIGN: Prescription register study. SETTING: Norwegian nationwide prescriptions socio-economic and disability status data. METHODS: Cox regression analyses. SUBJECTS: New benzodiazepine or Z-hypnotic users. MAIN OUTCOME MEASURE: Time to receive disability pension given benzodiazepine or Z-hypnotic use or both. Additional analyses focused on the benzodiazepine first redeemed. RESULTS: Among new users 8.65% of Z-hypnotic users, 12.29% of benzodiazepines users and 13.96% of combined Z-hypnotic and benzodiazepine users became disability pensioners. Z-hypnotic users were weaker associated with becoming disability pensioners (HR = 0.78, CI: 0.73-0.84) and combined users were stronger associated (HR = 1.09, CI: 1.01-1.17), than benzodiazepine users. Women had higher risk than men for becoming disability pensioners. Higher age, lower education, previous drug use and psychiatrist as first prescriber were risk factors. Comparing first benzodiazepine redeemed; clonazepam initiators were stronger associated with becoming disability pensioners than diazepam initiators were (HR = 2.22, CI: 1.81-2.71). No differences between other benzodiazepine users were found. CONCLUSIONS: Adjusting for known risk factors gave lower risk for Z-hypnotic users compared to benzodiazepine users for receiving disability pension. Combined use increased the risk further. Clonazepam initiators are especially at risk. These findings may be helpful in prescribing situations to identify and guide individuals at risk for becoming disability pensioners.

CaMKII and at least two unidentified kinases phosphorylate regulatory light chain in non-contracting cardiomyocytes.

In cardiac tissue, regulatory light chain (RLC, myosin light chain 2) phosphorylation (Ser(15)) leads to modulation of muscle contraction through Ca(2+)-sensitization. To elucidate which kinases that are involved in the basal (diastolic phase) RLC phosphorylation, we studied non-contracting adult rat cardiomyocytes. RLC kinase activities in situ were unmasked by maximally inhibiting myosin light chain phosphatase (MLCP) by calyculin A in the absence and presence of various protein kinase inhibitors. Surprisingly MLCK did not contribute to the phosphorylation of RLC in the non-contracting cardiomyocytes. Two kinase activity groups were revealed by different sensitivities to staurosporine. The fraction with the highest sensitivity to staurosporine was inhibited by KN-93, a selective CaMKII inhibitor, producing a 23% ± 7% reduction in RLC phosphorylation. Calmodulin antagonism (W7) and reduction in Ca(2+) (EGTA) combined with low concentration of staurosporine caused a larger decrease in RLC phosphorylation than staurosporine alone. These data strongly suggest that in addition to CaMKII, there is another Ca(2+)/calmodulin-dependent kinase and a Ca(2+)/calmodulin-independent kinase phosphorylating RLC. Thus the RLC phosphorylation seems to be ensured by redundant kinase activities.

CaMKII in addition to MLCK contributes to phosphorylation of regulatory light chain in cardiomyocytes.

The aim was to identify kinase activities involved in the phosphorylation of regulatory light chain (RLC) in situ in cardiomyocytes. In electrically stimulated rat cardiomyocytes, phosphatase inhibition by calyculin A unmasked kinase activities evoking an increase of phosphorylated RLC (P-RLC) from about 16% to about 80% after 80 min. The phosphorylation rate in cardiomyocytes was reduced by about 40% by the myosin light chain kinase (MLCK) inhibitor, ML-7. In rat ventricular muscle strips, calyculin A induced a positive inotropic effect that correlated with P-RLC levels. The inotropic effect and P-RLC elevation were abolished by ML-7 treatment. The kinase activities phosphorylating RLC in cardiomyocytes were reduced by about 60% by the non-selective kinase inhibitor staurosporine and by about 50% by the calmodulin antagonist W7. W7 eliminated the inhibitory effect of ML-7, suggesting that the cardiac MLCK is Ca(2+)/calmodulin (CaM)-dependent. The CaM-dependent kinase II (CaMKII) inhibitor KN-93 attenuated the calyculin A-induced RLC phosphorylation by about 40%, indicating a contribution from CaMKII. The residual phosphorylation in the presence of W7 indicated that also CaM-independent kinase activities might contribute. RLC phosphorylation was insensitive to protein kinase C inhibition. In conclusion, in addition to MLCK, CaMKII phosphorylates RLC in cardiomyocytes. Involvement of other kinases cannot be excluded.

Risk factors for excessive benzodiazepine use in a working age population: a nationwide 5-year survey in Norway.

OBJECTIVE: To identify risk factors for becoming an excessive user over time. SETTING: Prescription database study over five years. SUBJECTS AND METHOD: Norwegians between 30 and 60 years with a first dispensation of a benzodiazepine during 2006, encompassing 23 227 individuals. A Cox hazard regression model was defined, initially stratifying on gender, age, county, previous relevant drug dispensations, household income, education level, and vocational rehabilitation support. MAIN OUTCOME MEASURE: The time from the first redemption until excessive use was defined as using more than two DDDs per day on average within a three-month period. RESULTS: Women's risk was lower than men's for excessive use (HR = 0.42, CI 0.35-0.51). Initial oxazepam, alprazolam, or nitrazepam/flunitrazepam use indicated higher risk compared with diazepam (HR = 1.51, CI 1.24-1.85, HR = 2.75, CI 1.54-4.91, HR = 1.67, CI 1.29-2.16). Previous antidepressants or lithium, antipsychotics or opioids, anti-alcohol and smoke cessation treatment indicated a higher risk compared with no such use (HR = 1.4, CI 1.16-1.69, HR = 1.92, CI 1.54-2.4, and HR = 2.88, CI 2-4.15). Higher education and average or high household income were associated with a low risk compared with low education and income (HR = 0.68, CI 0.57-0.81, HR = 0.58, CI 0.46-0.73, and HR = 0.37, CI 0.26-0.54). Working in the private or public sector was associated with a low risk compared with no registered work (HR = 0.53, CI 0.4-0.71 and HR = 0.57, CI 0.45-0.74). CONCLUSION: The prevalence of excessive use over a five-year observation period was 2.34%. Risk factors were indications of psychiatric illness, first benzodiazepine choice, low income, and education. Excessive users were also characterized by a more severe disease, indicated by having prescription fulfilments by a psychiatrist and by switching benzodiazepines. Key points Guidelines state that benzodiazepines should be used for a short time and excessive use indicates drug dependency. Of all new benzodiazepine users 2.34% became excessive users, defined as consuming above two defined daily doses (DDDs) per day on average over three months, within a five-year period. Previous use of other psychotropic drugs, opioids and anti-alcohol and smoke cessation drugs, first benzodiazepine prescribed, low household income, and low education were risk factors for excessive use. Excessive users were characterized by switching benzodiazepines and having prescription fulfilments by a psychiatrist suggesting a more severe disease.

Different compartmentation of responses to brain natriuretic peptide and C-type natriuretic peptide in failing rat ventricle.

We previously found a negative inotropic (NIR) and positive lusitropic response (LR) to C-type natriuretic peptide (CNP) in the failing heart ventricle. In this study, we investigated and compared the functional responses to the natriuretic peptides (NPs), brain (BNP) and C-type natriuretic peptide (CNP), and relate them to cGMP regulation and effects on downstream targets. Experiments were conducted in left ventricular muscle strips and ventricular cardiomyocytes from Wistar rats with heart failure 6 weeks after myocardial infarction. As opposed to CNP, BNP did not cause an NIR or LR, despite increasing cGMP levels. The BNP-induced cGMP elevation was mainly and markedly regulated by phosphodiesterase (PDE) 2 and was only marginally increased by PDE3 or PDE5 inhibition. Combined PDE2, -3, and -5 inhibition failed to reveal any functional responses to BNP, despite an extensive cGMP elevation. BNP decreased, whereas CNP increased, the amplitude of the Ca(2+) transient. BNP did not increase phospholamban (PLB) or troponin I (TnI) phosphorylation, Ca(2+) extrusion rate constant, or sarcoplasmatic reticulum Ca(2+) load, whereas CNP did. Both BNP and CNP reduced the peak of the L-type Ca(2+) current. Cyclic GMP elevations by BNP and CNP in cardiomyocytes were additive, and the presence of BNP did not alter the NIR to CNP or the CNP-induced PLB and TnI phosphorylation. However, a small increase in the LR to maximal CNP was observed in the presence of BNP. In conclusion, different responses to cGMP generated by BNP and CNP suggest different compartmentation of the cGMP signal and different roles of the two NPs in the failing heart.

Connective tissue growth factor/CCN2 attenuates ß-adrenergic receptor responsiveness and cardiotoxicity by induction of G protein-coupled receptor kinase-5 in cardiomyocytes.

Myocardial connective tissue growth factor (CTGF/CCN2) is induced in heart failure, a condition associated with diminution of ß-adrenergic receptor (ß-AR) responsiveness. Accordingly, we aimed to investigate whether CTGF could play a mechanistic role in regulation of ß-AR responsiveness. Concentration-response curves of isoproterenol-stimulated cAMP generation in cardiomyocytes from transgenic mice with cardiac-restricted overexpression of CTGF (Tg-CTGF) or cardiomyocytes pretreated with recombinant human CTGF (rec-hCTGF) revealed marked reduction of both ß1-AR and ß2-AR responsiveness. Consistently, ventricular muscle strips from Tg-CTGF mice stimulated with isoproterenol displayed attenuation of maximal inotropic responses. However, no differences of maximal inotropic responses of myocardial fibers from Tg-CTGF mice and nontransgenic littermate control (NLC) mice were discerned when stimulated with supramaximal concentrations of dibutyryl-cAMP, indicating preserved downstream responsiveness to cAMP. Congruent with a mechanism of desensitization of ß-ARs, mRNA and protein levels of G protein-coupled receptor kinase 5 (GRK5) were found isoform-selective upregulated in both cardiomyocytes from Tg-CTGF mice and cardiomyocytes exposed to rec-hCTGF. Corroborating a mechanism of GRK5 in CTGF-mediated control of ß-AR sensitivity, Chinese hamster ovary cells pretreated with rec-hCTGF displayed increased agonist- and biased ligand-stimulated ß-arrestin binding to ß-ARs. Despite increased sensitivity of cardiomyocytes from GRK5-knockout (KO) mice to ß-adrenergic agonists, pretreatment of GRK5-KO cardiomyocytes with rec-hCTGF, as opposed to cardiomyocytes from wild-type mice, did not alter ß-AR responsiveness. Finally, Tg-CTGF mice subjected to chronic exposure (14 days) to isoproterenol revealed blunted myocardial hypertrophy and preserved cardiac function versus NLC mice. In conclusion, this study uncovers a novel mechanism controlling ß-AR responsiveness in cardiomyocytes involving CTGF-mediated regulation of GRK5.

Activation of Liver X receptors in the heart leads to accumulation of intracellular lipids and attenuation of ischemia-reperfusion injury.

Liver X receptor (LXR)-α and -ß play a major role in lipid and glucose homeostasis. Their expression and function in the heart is not well characterized. Our aim was to describe the expression of LXRs in the murine heart, and to determine effects of cardiac LXR activation on target gene expression, lipid homeostasis and ischemia. Both LXRα and -ß were expressed in heart tissues, HL-1 cells and isolated cardiomyocytes as determined by qRT-PCR. Elevated cardiac expression of LXR target genes and LXRß was observed 24 h after in vivo permanent coronary artery ligation. The synthetic LXR agonist GW3965 induced mRNA expression of the LXR target genes in HL-1 cells and isolated cardiomyocytes. This was associated with a buildup of intracellular triglycerides and expanding lipid droplets as quantified by confocal microscopy. Mice injected with GW3965 had cardiac LXR activation as judged by increased target gene expression and lipid droplet accumulation. GW3965 in vivo and in vitro increased expression of genes inducing triglyceride synthesis, and altered expression of lipid droplet-binding protein genes. GW3965 protected HL-1 cells against hypoxia-reoxygenation induced apoptosis. LXR activation by GW3965 in vivo prior to heart isolation and perfusion with induced global ischemia and reperfusion improved left ventricular contractile function and decreased infarct size. In conclusion, LXRs are expressed in the murine heart in the basal state, and are activated by myocardial infarction. Activation of LXR by the synthetic agonist GW3965 is associated with intracardiac accumulation of lipid droplets and protection against myocardial ischemia-reperfusion injury.

[Dispensing of benzodiazepines and Z drugs by Norwegian pharmacies 2004-2011]. / Utlevering av benzodiazepiner og z-hypnotika fra norske apotek 2004-11.

BACKGROUND: In Norway, total sales of benzodiazepines and Z drugs (zopiclone and zolpidem) have increased since the mid-1990s. On the basis of data from the Norwegian Prescription Database, we have studied the choice of medications and patterns of use in various gender and age groups. MATERIAL AND METHOD: Numbers for redemptions of benzodiazepines and Z drugs in Norwegian pharmacies for the years 2004-2011 were collected from the Prescription Registry. Population figures were collected from Statistics Norway. Consumption was calculated by the number of DDDs (defined daily doses). RESULTS: Among those who were supplied with benzodiazepines or Z drugs, recipients of more than 2 DDDs per day (heavy users) accounted for a small group. We registered an extensive use of Z drugs among older women, many of whom were prescribed an amount corresponding to a regular daily use of 1-2 DDDs. The total prescription of alprazolam and nitrazepam/flunitrazepam was minor, but high dosages were not uncommon among those who were prescribed these drugs. Only a small proportion of the patients who were prescribed clonazepam received a reimbursable prescription. The prescribing of a number of benzodiazepines and Z drugs at the same time remains not uncommon. INTERPRETATION: Prescribing of Z drugs to the elderly, and to women in particular, may indicate that many people in this group are regular users of sedative hypnotics, which is contrary to the guidelines. A considerable proportion of the prescriptions for clonazepam are outside of the approved indications. Among the users of the drugs studied, only a small fraction were heavy users, but since the use is widespread, this represents a considerable number of individuals.

Prostaglandin E1 facilitates inotropic effects of 5-HT4 serotonin receptors and ß-adrenoceptors in failing human heart.

Prostaglandins have displayed both beneficial and detrimental effects in clinical studies in patients with severe heart failure. Prostaglandins are known to increase cardiac output, but the mechanism is not clarified. Here, we tested the hypothesis that prostaglandins can increase contractility in human heart by amplifying cAMP-dependent inotropic responses. Contractility was measured ex vivo in isolated left ventricular strips and phosphodiesterase (PDE) and adenylyl cyclase (AC) activity was measured in homogenates or membranes from failing human left ventricles. PGE(1) (1 µM) alone did not modify contractility, but given prior, amplified maximal serotonin (5-HT)-evoked (10 µM) contractile responses mediated by 5-HT(4) receptors several fold (24 ± 7 % with PGE(1) vs. 3 ± 2 % above basal with 5-HT alone). The 5-HT(4)-mediated inotropic response was amplified by the PDE3 inhibitor cilostamide and further amplified in combination with PGE(1) (26 ± 6 vs. 56 ± 12 % above basal). PGE(1) reduced the time to reach 90 % of both the maximal 5-HT- and isoproterenol-evoked inotropic response compared to 5-HT or isoproterenol alone. PGE(1) did not modify PDE activity in the homogenate, either alone or when given simultaneously with PDE3 and/or PDE4 inhibitors. Neither 5-HT- nor isoproterenol-stimulated AC activity was significantly amplified by PGE(1). Sensitivity of ventricular strips to Ca(2+) was not enhanced in the presence of PGE(1). Our results show that PGE(1) can enhance cAMP-mediated responses in failing human left ventricle, through a mechanism independent of PDE inhibition, amplification of AC activity or increasing sensitivity to calcium. This effect of PGE(1) possibly contributes to the increase of cardiac output, independent of decreased afterload, observed after prostaglandin administration in humans.

A review and Bayesian meta-analysis of clinical efficacy and adverse effects of 4 atypical neuroleptic drugs compared with haloperidol and placebo.

AIMS: The objective of the study was to examine the efficacy and the degree of adverse effects connected with atypical neuroleptic drugs and haloperidol by using a previously described Bayesian statistical method that includes both direct and indirect comparisons simultaneously. METHODS: The authors used the results of 30 double-blind, randomized studies including comparisons of 4 atypical neuroleptics and haloperidol, head-to-head or against placebo. We calculated the response ratios for drugs against placebo and thereafter the relative response ratios for one drug against another. With uniform priors, we calculated and ranked the posterior estimates of response ratios for antipsychotic effect, weight gain, and occurrence of extrapyramidal symptoms. RESULTS: All second-generation neuroleptics analyzed are fairly effective with response ratios against placebo ranging between 1.55 (credibility interval, 1.36-1.76) and 1.99 (1.76-2.26), with clozapine being the most effective and aripiprazole the least effective among them. The risk of inducing weight gain is clearly very high for all 5 neuroleptic drugs compared with placebo with response ratios of 12.21 (10.22-15.05) for olanzapine and 11.28 (6.89-17.77) for clozapine. There is a clear increased risk of extrapyramidal adverse effects for haloperidol compared with placebo as the response ratio is 2.33 (2.03-2.49). The other drugs all have considerably less risk of extrapyramidal adverse effects. CONCLUSIONS: The 4 second-generation neuroleptics included in our meta-analysis show only small differences in overall efficacy, with clozapine being the most effective and aripiprazole the least effective among them. When the risk of adverse effects is analyzed, olanzapine and clozapine are afflicted with the highest risk of inducing weight gain and haloperidol with extrapyramidal symptoms. Even aripiprazole and risperidone, however, induce considerable weight gain compared with placebo but may be acceptable alternatives when tailoring drug treatment to the individual patient.

Constitutive inhibitory G protein activity upon adenylyl cyclase-dependent cardiac contractility is limited to adenylyl cyclase type 6.

PURPOSE: We previously reported that inhibitory G protein (Gi) exerts intrinsic receptor-independent inhibitory activity upon adenylyl cyclase (AC) that regulates contractile force in rat ventricle. The two major subtypes of AC in the heart are AC5 and AC6. The aim of this study was to determine if this intrinsic Gi inhibition regulating contractile force is AC subtype selective. METHODS: Wild-type (WT), AC5 knockout (AC5KO) and AC6 knockout (AC6KO) mice were injected with pertussis toxin (PTX) to inactivate Gi or saline (control).Three days after injection, we evaluated the effect of simultaneous inhibition of phosphodiesterases (PDE) 3 and 4 with cilostamide and rolipram respectively upon in vivo and ex vivo left ventricular (LV) contractile function. Also, changes in the level of cAMP were measured in left ventricular homogenates and at the membrane surface in cardiomyocytes obtained from the same mouse strains expressing the cAMP sensor pmEPAC1 using fluorescence resonance energy transfer (FRET). RESULTS: Simultaneous PDE3 and PDE4 inhibition increased in vivo and ex vivo rate of LV contractility only in PTX-treated WT and AC5KO mice but not in saline-treated controls. Likewise, Simultaneous PDE3 and PDE4 inhibition elevated total cAMP levels in PTX-treated WT and AC5KO mice compared to saline-treated controls. In contrast, simultaneous PDE3 and PDE4 inhibition did not increase in vivo or ex vivo rate of LV contractility or cAMP levels in PTX-treated AC6KO mice compared to saline-treated controls. Using FRET analysis, an increase of cAMP level was detected at the membrane of cardiomyocytes after simultaneous PDE3 and PDE4 inhibition in WT and AC5KO but not AC6KO. These FRET data are consistent with the functional data indicating that AC6 activity and PTX inhibition of Gi is necessary for simultaneous inhibition of PDE3 and PDE4 to elicit an increase in contractility. CONCLUSIONS: Together, these data suggest that AC6 is tightly regulated by intrinsic receptor-independent Gi activity, thus providing a mechanism for maintaining low basal cAMP levels in the functional compartment that regulates contractility.

Effects of serotonin in failing cardiac ventricle: signalling mechanisms and potential therapeutic implications.

Previously, cardioexcitation by serotonin (5-hydroxytryptamine, 5-HT) was believed to be confined to atria in mammals including man, and mediated through 5-HT(4) receptors in pig and man, but 5-HT(2A) receptors in rat. Recent studies, reviewed here, demonstrate that functional 5-HT(4) receptors can be revealed in porcine and human ventricular myocardium during phosphodiesterase inhibition, and that 5-HT(4) receptor mRNA is increased in human heart failure. In rats, functional 5-HT(4) and 5-HT(2A) receptors appear in the cardiac ventricle during heart failure and mediate inotropic responses through different mechanisms. 5-HT(2A) receptor signalling resembles that from alpha(1)-adrenoceptors and causes inotropic effects through increased myosin light chain phosphorylation, resulting in Ca(2+) sensitisation. 5-HT(4) receptor signalling resembles that from beta-adrenoceptors and causes inotropic effects through a pathway involving cAMP and PKA-mediated phosphorylation of proteins involved in Ca(2+) handling, resulting in enhanced contractility through increased Ca(2+) availability. Cyclic AMP generated through 5-HT(4) receptor stimulation seems more efficiently coupled to increased contractility than cAMP generated through beta-adrenoceptor stimulation. Increasing contractility through cAMP is considered less energy efficient than Ca(2+) sensitisation and this may be one reason why beta-adrenoceptor antagonism is beneficial in heart failure patients. Treatment of heart failure rats with the 5-HT(4) antagonist SB207266 (piboserod) resulted in potentially beneficial effects, although small. Further studies are needed to clarify if such treatment will be useful for patients with heart failure.

Calculating reaction rate constants and estimating the efficacy of selective enzyme inhibitors.

The dynamic steady state of a pair of forward and backward enzymatic reactions is dependent on the balance between the enzymes catalyzing the reactions. By selectively inhibiting one or more of the enzymes involved, this balance is shifted into a new steady state, making it possible to calculate the reaction rate constants after measurement of the reactants. Ideally, the inhibitors should completely eliminate either reaction, but this is often not the case. Here we present and discuss a method for calculating the reaction rate constants and, thus, for evaluating the efficacy of one or more inhibitors when introduced to a forward-backward pair of enzymatic reactions.

Pro-inflammatory potential of ultrafine particles in mono- and co-cultures of primary cardiac cells.

Inhalation of particulate air pollution has been associated with increased risks for cardiovascular mortality and morbidity, but the underlying mechanisms are still under discussion. One possible pathway may be that inhaled particles cross the air-blood barrier and interact directly with cardiac tissue. The aim of the present study was to examine the pro-inflammatory potential of particles in cardiac cells. Mono- and co-cultures of primary adult male Wistar (Han) rat cardiomyocytes (CMs) and cardiofibroblasts (CFs) were exposed to increasing concentrations of ultrafine (<100nm) carbon black particles (Printex 90). Expression and release of cytokines (IL-6, IL-1beta and TNF-alpha) were measured by using quantitative real-time PCR and ELISA, respectively. Cytotoxicity was estimated by measuring cellular release of lactate dehydrogenase (LDH). A particle concentration-dependent increase in IL-6 release was observed in both CM mono- and co-cultures (EC(50) approximately 57microg/ml). Furthermore, IL-6 levels detected in both control and particle-exposed co-cultures were synergistically increased compared to mono-cultures (10-19-fold, dependent on the exposure). Experiments with contact and non-contact co-cultures indicate that direct cellular contact is of key importance for the enhanced release of IL-6 in co-cultures. An apparent particle-induced release of IL-1beta was only detected in co-cultures. The release of TNF-alpha was low and did not seem notably influenced by particle exposure. Treatment with an IL-1 receptor antagonist apparently eliminated the particle-induced release of IL-6. In conclusion, ultrafine particles have a pro-inflammatory potential in primary cardiac cells. Furthermore, IL-1 seems critical in triggering particle-induced release of IL-6. These pro-inflammatory responses may be elicited when particles are translocated into the pulmonary circulation upon inhalation or administered intravascularly during medical procedures.

Mental Health and Disability Pension Onset: Changes in Consumption of Antianxiety and Hypnotic Drugs.

INTRODUCTION: In Norway, disability pension (DP) has been more prevalent over the later years, with mental disorders being a frequent cause. Previous analyses have questioned whether receiving DP is beneficial for mental health by considering changes in antidepressant drug consumption. To explore this further, we examined changes in antianxiety and hypnotic drug consumption following DP onset. METHODS: Based on national Norwegian register data, this retrospective study encompassed 8617 working-age individuals (25-50 years) who became DP during 2005 to 2013. We compared their benzodiazepines (BZD) and Z-hypnotic consumption 1 year pre- and postdisability pension onset. RESULTS: About 80% of the individuals did not change their altogether benzodiazepine/Z-hypnotic consumption. Among individuals with an initial consumption ≤1 defined daily dose (DDD), 18.9% increased their consumption to above 1 DDD. Individuals in the age-group 45 to 50 versus 24 to 34 years had a lower risk of dose escalation (odds ratio [OR], 0.756, 95% confidence interval [CI]: 0.601-0.957). Individuals who used Z-hypnotics only had a higher risk of dose escalation compared to the joint benzodiazepine/Z-hypnotic user group (OR, 1.594, 95%CI: 1.284-1.970). CONCLUSION: In general, we cannot see that DP is associated with changes in benzodiazepine/Z-hypnotic consumption, but younger users and individuals using Z-hypnotics only had a greater risk of dose escalation compared to the older users and users with combined BZD and Z-hypnotic use.

Dual serotonergic regulation of ventricular contractile force through 5-HT2A and 5-HT4 receptors induced in the acute failing heart.

Cardiac responsiveness to neurohumoral stimulation is altered in congestive heart failure (CHF). In chronic CHF, the left ventricle has become sensitive to serotonin because of appearance of Gs-coupled 5-HT4 receptors. Whether this also occurs in acute CHF is unknown. Serotonin responsiveness may develop gradually or represent an early response to the insult. Furthermore, serotonin receptor expression could vary with progression of the disease. Postinfarction CHF was induced in male Wistar rats by coronary artery ligation with nonligated sham-operated rats as control. Contractility was measured in left ventricular papillary muscles and mRNA quantified by real-time reverse-transcription PCR. Myosin light chain-2 phosphorylation was determined by charged gel electrophoresis and Western blotting. Ca2+ transients in CHF were measured in field stimulated fluo-4-loaded cardiomyocytes. A novel 5-HT2A receptor-mediated inotropic response was detected in acute failing ventricle, accompanied by increased 5-HT2A mRNA levels. Functionally, this receptor dominated over 5-HT4 receptors that were also induced. The 5-HT2A receptor-mediated inotropic response displayed a triphasic pattern, shaped by temporally different activation of Ca2+-calmodulin-dependent myosin light chain kinase, Rho-associated kinase and inhibitory protein kinase C, and was accompanied by increased myosin light chain-2 phosphorylation. Ca2+ transients were slightly decreased by 5-HT2A stimulation. The acute failing rat ventricle is, thus, dually regulated by serotonin through Gq-coupled 5-HT2A receptors and Gs-coupled 5-HT4 receptors.

PDE3 inhibition by C-type natriuretic peptide-induced cGMP enhances cAMP-mediated signaling in both non-failing and failing hearts.

We have previously shown that the natriuretic peptide receptor B (NPR-B) agonist C-type natriuretic peptide (CNP) enhances cyclic adenosine 3´,5´-monophosphate (cAMP)-mediated signaling in failing hearts, through cyclic guanosine 3´,5´-monophosphate (cGMP)-mediated phosphodiesterase (PDE) 3 inhibition. As several signaling pathways are importantly changed in failing hearts, it could not be taken for granted that this crosstalk would be the same in non-failing hearts. Thus, we wanted to clarify to which extent this effect of CNP occurred also in non-failing hearts. Inotropic and lusitropic responses were measured in muscle strips and cGMP levels, localized cAMP levels, cAMP-PDE activity and mRNA levels were analyzed in isolated cardiomyocytes from left ventricles of non-failing and failing rat hearts. CNP increased cGMP and enhanced ß1- and ß2-adrenoceptor-mediated inotropic and ß1-adrenoceptor-mediated lusitropic responses, in non-failing and failing hearts. The NPR-A agonist brain natriuretic peptide (BNP) increased cGMP, but did not affect inotropic or lusitropic responses, indicating different compartmentation of cGMP from the two natriuretic peptide receptors. cAMP-PDE activity of PDE3 was concentration-dependently inhibited by cGMP with the same potency and to the same extent in non-failing and failing cardiomyocytes. CNP enhanced ß1-adrenoceptor-induced cAMP increase in living cardiomyocytes in the absence, but not in the presence of a PDE3 inhibitor indicating involvement of PDE3. In summary, CNP sensitizes cAMP-mediated signaling in non-failing as in failing hearts, via NPR-B-mediated increase of cGMP that inhibits the cAMP-PDE activity of PDE3.