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.

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.

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.

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.

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.

A 5-year follow-up study of users of benzodiazepine: starting with diazepam versus oxazepam.

BACKGROUND: Drug dependency may develop during long-term benzodiazepine use, indicated, for example, by dose escalation. The first benzodiazepine chosen may affect the risk of dose escalation. AIM: To detect possible differences in benzodiazepine use between new users of diazepam and oxazepam over time. DESIGN AND SETTING: This 5-year prescription database study included 19 747 new benzodiazepine users, inhabitants of Norway, aged 30-60 years, with first redemption for diazepam or oxazepam. METHOD: Individuals starting on diazepam versus oxazepam were analysed by logistic regression with sex, age, other drug redemptions, prescriber's specialty, household income, education level, type of work, and vocational rehabilitation support as background variables. Time to reach a daily average intake of ≥1 defined daily doses (DDD) over a 3-month period was analysed using a Cox proportional hazard regression model. RESULTS: New users of oxazepam had a higher risk for dose escalation compared with new users of diazepam. This was true even when accounting for differences in sociodemographic status and previous drug use (hazard ratio [HR] 1.33, 95% confidence interval = 1.17 to 1.51). CONCLUSION: Most doctors prescribed, according to recommendations, oxazepam to individuals they may have regarded as prone to and at risk of dependency. However, these individuals were at higher risk for dose escalation even when accounting for differences in sociodemographic status and previous drug use. Differences between the two user groups could be explained by different preferences for starting drug, DDD for oxazepam being possibly too low, and some unaccounted differences in illness.

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.

The inotropic effect of the active metabolite of levosimendan, OR-1896, is mediated through inhibition of PDE3 in rat ventricular myocardium.

AIMS: We recently published that the positive inotropic response (PIR) to levosimendan can be fully accounted for by phosphodiesterase (PDE) inhibition in both failing human heart and normal rat heart. To determine if the PIR of the active metabolite OR-1896, an important mediator of the long-term clinical effects of levosimendan, also results from PDE3 inhibition, we compared the effects of OR-1896, a representative Ca2+ sensitizer EMD57033 (EMD), levosimendan and other PDE inhibitors. METHODS: Contractile force was measured in rat ventricular strips. PDE assay was conducted on rat ventricular homogenate. cAMP was measured using RII_epac FRET-based sensors. RESULTS: OR-1896 evoked a maximum PIR of 33 ± 10% above basal at 1 µM. This response was amplified in the presence of the PDE4 inhibitor rolipram (89 ± 14%) and absent in the presence of the PDE3 inhibitors cilostamide (0.5 ± 5.3%) or milrinone (3.2 ± 4.4%). The PIR was accompanied by a lusitropic response, and both were reversed by muscarinic receptor stimulation with carbachol and absent in the presence of ß-AR blockade with timolol. OR-1896 inhibited PDE activity and increased cAMP levels at concentrations giving PIRs. OR-1896 did not sensitize the concentration-response relationship to extracellular Ca2+. Levosimendan, OR-1896 and EMD all increased the sensitivity to ß-AR stimulation. The combination of either EMD and levosimendan or EMD and OR-1896 further sensitized the response, indicating at least two different mechanisms responsible for the sensitization. Only EMD sensitized the α1-AR response. CONCLUSION: The observed PIR to OR-1896 in rat ventricular strips is mediated through PDE3 inhibition, enhancing cAMP-mediated effects. These results further reinforce our previous finding that Ca2+ sensitization does not play a significant role in the inotropic (and lusitropic) effect of levosimendan, nor of its main metabolite OR-1896.

Non-classical regulation of ß1- and ß 2-adrenoceptor-mediated inotropic responses in rat heart ventricle by the G protein Gi.

Studies suggest that increased activity of Gi contributes to the reduced ß-adrenoceptor-mediated inotropic response (ßAR-IR) in failing cardiomyocytes and that ß2AR-IR but not ß1AR-IR is blunted by dual coupling to Gs and Gi. We aimed to clarify the role of Gi upon the ß1AR-IR and ß2AR-IR in Sham and failing myocardium by directly measuring contractile force and cAMP accumulation. Contractility was measured ex vivo in left ventricular strips and cAMP accumulation in cardiomyocytes from rats with post-infarction heart failure (HF) or sham operates (Sham). The ß2AR-IR in Sham and HF was small and was amplified by simultaneously inhibiting phosphodiesterases 3 and 4 (PDE3&4). In HF, the inotropic response and cAMP accumulation evoked by ß1AR- or ß2AR-stimulation were reduced. Inactivation of Gi with pertussis toxin (PTX) did not restore the ß1AR-IR or ß2AR-IR in HF to Sham levels but did enhance the maximal ß2AR-IR. PTX increased both ß1AR- and ß2AR-evoked cAMP accumulation more in Sham than that in HF, and HF levels approached those in untreated Sham. The potency of agonists at ß1 and at ß2ARs (only under PDE3&4 inhibition) was increased in HF and by PTX in both HF and Sham. Without PDE3&4 inhibition, PTX increased only the maximal ß2AR-IR, not potency. We conclude that Gi regulates both ß1AR- and ß2AR-IR independent of receptor coupling with Gi. Gi together with PDE3&4 tonically restrict the ß2AR-IR. Gi inhibition did not restore the ßAR-IR in HF despite increasing cAMP levels, suggesting that the mechanism of impairment resides downstream to cAMP signalling.

Gi proteins regulate adenylyl cyclase activity independent of receptor activation.

BACKGROUND AND PURPOSE: Despite the view that only ß2- as opposed to ß1-adrenoceptors (ßARs) couple to G(i), some data indicate that the ß1AR-evoked inotropic response is also influenced by the inhibition of Gi. Therefore, we wanted to determine if Gi exerts tonic receptor-independent inhibition upon basal adenylyl cyclase (AC) activity in cardiomyocytes. EXPERIMENTAL APPROACH: We used the Gs-selective (R,R)- and the Gs- and G(i)-activating (R,S)-fenoterol to selectively activate ß2ARs (ß1AR blockade present) in combination with Gi inactivation with pertussis toxin (PTX). We also determined the effect of PTX upon basal and forskolin-mediated responses. Contractility was measured ex vivo in left ventricular strips and cAMP accumulation was measured in isolated ventricular cardiomyocytes from adult Wistar rats. KEY RESULTS: PTX amplified both the (R,R)- and (R,S)-fenoterol-evoked maximal inotropic response and concentration-dependent increases in cAMP accumulation. The EC50 values of fenoterol matched published binding affinities. The PTX enhancement of the Gs-selective (R,R)-fenoterol-mediated responses suggests that Gi regulates AC activity independent of receptor coupling to Gi protein. Consistent with this hypothesis, forskolin-evoked cAMP accumulation was increased and inotropic responses to forskolin were potentiated by PTX treatment. In non-PTX-treated tissue, phosphodiesterase (PDE) 3 and 4 inhibition or removal of either constitutive muscarinic receptor activation of Gi with atropine or removal of constitutive adenosine receptor activation with CGS 15943 had no effect upon contractility. However, in PTX-treated tissue, PDE3 and 4 inhibition alone increased basal levels of cAMP and accordingly evoked a large inotropic response. CONCLUSIONS AND IMPLICATIONS: Together, these data indicate that Gi exerts intrinsic receptor-independent inhibitory activity upon AC. We propose that PTX treatment shifts the balance of intrinsic G(i) and Gs activity upon AC towards Gs, enhancing the effect of all cAMP-mediated inotropic agents.

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.

Differential regulation of C-type natriuretic peptide-induced cGMP and functional responses by PDE2 and PDE3 in failing myocardium.

Recently, we showed C-type natriuretic peptide (CNP)-induced negative inotropic (NIR) and positive lusitropic response (LR) in failing rat heart. We wanted to study whether, and if so, how phosphodiesterases (PDEs) regulate CNP-induced cyclic 3',5'-guanosine monophosphate (cGMP) elevation and functional responses. Inotropic and lusitropic responses were measured in left ventricular muscle strips and cyclic nucleotide levels, PDE activity and phospholamban (PLB) and troponin I (TnI) phosphorylation were measured in ventricular cardiomyocytes from Wistar rats with heart failure 6 weeks after myocardial infarction. CNP-mediated increase in global cGMP was mainly regulated by PDE2, as reflected by a marked amplification of the cGMP increase during PDE2 inhibition and by a high PDE2 activity in cardiomyocytes. PDE3 inhibition, on the other hand, caused no significant cGMP increase by CNP. The functional consequences did not correspond to the changes of cGMP. PDE3 inhibition increased the potency of the CNP-induced NIR and LR, while PDE2 inhibition desensitized the CNP-induced NIR, but not LR. A role for PDE2 on the maximal LR and PDE5 on the maximal NIR to CNP was revealed in the presence of PDE3 inhibition. CNP increased PLB phosphorylation about 25- to 30-fold and tended to increase TnI phosphorylation about twofold. As a whole, CNP-induced functional responses were only modestly regulated by PDEs compared to the cAMP-mediated functional responses to ß1-adrenoceptor stimulation, which are highly regulated by PDEs. There is a mismatch between the CNP-induced cGMP increase and functional responses. Global cGMP levels are mainly regulated by PDE2 after CNP stimulation, whereas the functional responses are modestly regulated by both PDE2 and PDE3, indicating cGMP compartmentation by PDEs affecting CNP-induced responses in failing hearts.

Identification of small molecule NPR-B antagonists by high throughput screening--potential use in heart failure.

We found previously that stimulation of natriuretic peptide receptor (NPR)-B by C-type natriuretic peptide (CNP) in failing rat ventricle potentiates ß1-adrenoceptor (ß1-AR)-mediated inotropic response to noradrenaline through cGMP-mediated inhibition of phosphodiesterase (PDE) 3, thereby enhancing cAMP-mediated signalling. Increased cAMP-mediated signalling is deleterious in chronic heart failure (HF; basis for the use of ß-blockers in HF) and we propose to consider NPR-B antagonists as new HF treatment in addition to conventional therapy. Since there is no NPR-B-selective antagonist available for clinical studies, we aimed at identifying a novel small molecule (non-peptide) NPR-B antagonist. An assay was developed and high throughput screening performed on a chemical library of about 20,000 small molecule compounds (<500 Da) to identify NPR-B antagonists based on inhibition of CNP-stimulated cGMP production in NPR-B-expressing HEK293 cells. The screen revealed several potential NPR-B antagonists, of which six were selected for further studies. Three showed selective NPR-B vs NPR-A inhibition and three were partially selective. The compounds mediated reversible, noncompetitive inhibition and most likely act as allosteric modulators binding outside the agonist binding site of NPR-B. In rat ventricular muscle strips, the potentiating effect of CNP upon ß1-AR-evoked inotropic effects could be attenuated by at least one of these compounds. We identified several small molecule NPR-B antagonists by high throughput screening and show in a functional heart preparation that blocking NPR-B stimulation with a small molecule compound can reduce the potentiating effect of CNP on the ß1-AR-mediated inotropic response to noradrenaline.

[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.

A 3-year survey quantifying the risk of dose escalation of benzodiazepines and congeners to identify risk factors to aid doctors to more rationale prescribing.

OBJECTIVES: This study investigated and quantified risk factors of dose escalation, as an indication of drug misuse and dependency of benzodiazepines and congeners, among presumably drug naïve patients in the Norwegian drug prescription database, observed over 3 years. DESIGN: Observational study. SETTING: Prescription database study. PARTICIPANTS: We defined an excessive user as one redeeming more than two defined daily doses per day in 3 months. PRIMARY AND SECONDARY OUTCOME MEASURES: We examined the risk of excessive use over time and the effect of risk factors through multistate logistic regression and scenarios. RESULTS: Most of the 81 945 patients had zopiclone or zolpidem as the initial drug (63.8%), followed by diazepam (25.3%), oxazepam (6.1%), nitrazepam/flunitrazepam (2.9%), hydroxyzine/buspirone (1.6%) and alprazolam (0.3%). At any time 23% redeemed prescriptions, about 34% did not redeem any prescriptions beyond any 3-month period and 0.9% ended up as excessive users. Patients previously using drugs, such as opioids, antialcohol or smoke cessation treatment, had a higher risk to become excessive users compared to patients who had not. Patients whose first prescription was for oxazepam or nitrazepam/flunitrazepam had a higher risk of becoming an excessive user compared to those who started with diazepam. A specialist in general practice as the first-time prescriber was associated with a lower risk compared to doctors without specialty. CONCLUSIONS: Most benzodiazepine use occurred according to guidelines. Still, some experienced dose escalation over time, and risk factors were previous use of other psychotropic drugs, long time use, choice of first-time drug and prescriber's specialty. This could incite doctors to have a cessation plan when issuing first-time prescriptions.

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.