CONSUMPTION TREND OF OPIOIDS IN AMBULATORY PATIENTS IN ALBANIA 2014-2019

Objective: The aim of this paper is to evaluate 6‐year trends in community use of prescribed opioid analgesics in Albania, using the Anatomic Therapeutic Chemical Classification-Defined Daily Dose methodology. Methods: We collected the data from the database of the Health Insurance Institute (HII). Analysis of the data includes the period 2014–2019; also, we analyzed the data of import and domestic production of drugs, which represent the real consumption of drugs in the country. These data were subsequently involved in a comparative analysis of the utilization data according to the HII. Results: We report a 2.5 fold increase in opioid utilization over the study period. The maximal rise in consumption refers to fentanyl, oxycodone, and tramadol. Meanwhile, the maximal values of consumption refer to strong opiate, morphine. We note that around 30% of the consumption of this class flows out of the scheme. Furthermore, around 25% of consumption of morphine and oxycodone flows out of scheme too. Meanwhile, the tramadol consumption runs out of the scheme over 200%. Conclusions: The outcomes indicate that patients in Albania have low access to opioids medications mainly because of low opioids availability. A strong opiophobia among the population and the medical professionals constitutes one of the major impediments for efficient palliative care.

Circulating catecholamines partially regulate T-wave morphology but not heart rate variability during repeated umbilical cord occlusions in fetal sheep

Fetal heart rate (FHR) variability (FHRV) and ST segment morphology are potential clinical indices of fetal well-being during labor. β-Adrenergic stimulation by circulating catecholamines has been hypothesized to contribute to both FHRV and ST segment morphology during labor, but this has not been tested during brief repeated fetal hypoxemia that is characteristic of labor. Near-term fetal sheep (0.85 gestation) received propranolol (β-adrenergic blockade; n = 10) or saline ( n = 7) 30 min before being exposed to three 2-min complete umbilical cord occlusions (UCOs) separated by 3-min reperfusions. T/QRS ratio was calculated throughout UCOs and reperfusion periods, and measures of FHRV (RMSSD, SDNN, and STV) were calculated between UCOs. During the baseline period, before the start of UCOs, propranolol was associated with reduced FHR, SDNN, and STV but did not affect RMSSD or T/QRS ratio. UCOs were associated with rapid FHR decelerations and increased T/QRS ratio; propranolol significantly reduced FHR during UCOs and was associated with a slower rise in T/QRS ratio during the first UCOs, without affecting the maximal rise or T/QRS ratio during the second and third UCO. Between UCOs propranolol reduced FHR and T/QRS ratio but did not affect any measure of FHRV. These data demonstrate that circulating catecholamines do not contribute to FHRV during labor-like hypoxemia. Furthermore, circulating catecholamines did not contribute to the major rise in T/QRS ratio during labor-like hypoxemia but may regulate T/QRS ratio between brief hypoxemia.

Twenty-four hour pharmacokinetic relationships for intravenous vancomycin and novel urinary biomarkers of acute kidney injury in a rat model

Objectives To identify the pharmacokinetic (PK) and toxicodynamic (TD) relationship for vancomycin-induced kidney injury. Methods Male Sprague–Dawley rats received intravenous (iv) vancomycin. Doses ranging from 150 mg/kg/day to 400 mg/kg/day were administered as a single or twice-daily injection over 24 h (total protocol duration). Controls received iv saline. Plasma was sampled with up to eight samples in 24 h per rat. Twenty-four hour urine was collected and assayed for kidney injury molecule 1 (KIM-1), osteopontin and clusterin. Vancomycin in plasma was quantified via LC-MS/MS. PK analyses were conducted using Pmetrics for R. PK exposures during the first 24 h (i.e. AUC0–24h, Cmax 0–24h and Cmin 0–24h) were calculated. PK/TD relationships were assessed with Spearman’s rank coefficient (rs) and the best-fit mathematical model. Results PK/TD data were generated from 45 vancomycin-treated and 5 control rats. A two-compartment model fit the data well (Bayesian: observed versus predicted R2 = 0.97). Exposure–response relationships were found between AUC0–24h versus KIM-1 and osteopontin (R2 = 0.61 and 0.66) and Cmax 0–24h versus KIM-1 and osteopontin (R2 = 0.50 and 0.56) using a four-parameter Hill fit. Conversely, Cmin 0–24h was less predictive of KIM-1 and osteopontin (R2 = 0.46 and 0.53). A vancomycin AUC0–24h of 482.2 corresponded to a 90% of maximal rise in KIM-1. Conclusions Vancomycin-induced kidney injury as defined by urinary biomarkers is driven by vancomycin AUC or Cmax rather than Cmin. Further, an identified PK/TD target AUC0–24h of 482.2 mg·h/L may have direct relevance to human outcomes.

Modulation of Transient and Persistent Inward Currents by Activation of Protein Kinase C in Spinal Ventral Neurons of the Neonatal Rat

Neuronal excitability can be regulated through modulation of voltage threshold ( Vth). Previous studies suggested that this modulation could be mediated by modulation of transient sodium currents ( IT) and/or persistent inward current (PIC). Modulation of IT and PIC through activation of protein kinase C (PKC) has previously been described as a mechanism controlling neuronal excitability. We investigated modulation of IT and PIC by PKC in neonatal rat spinal ventral neurons. In whole cell voltage clamp, activation of PKC by application of 1-oleoyl-2-acetyl-sn-glycerol (OAG, 10–30 μM) resulted in 1) a reduction of IT amplitude by 33% accompanied an increase in half-width and a decrease in the maximal rise and decay rates of the IT; 2) a reduction of PIC amplitude by 49%, with a depolarization of PIC onset by 4.5 mV. Activation of PKC caused varied effects on Vth for eliciting IT, with an unchanged Vth or depolarized Vth being the most common effects. In current-clamp recordings, PKC activation produced a small but significant depolarization (2.0 mV) of Vth for action potential generation with an increase in half-width and a decrease in amplitude and the maximal rise and decay rates of action potentials. Inclusion of PKCI19–36 (10–30 μM), a PKC inhibitor, in the recording pipette could block the OAG effects on IT and PIC. The ability of serotonin to hyperpolarize Vth was not altered by PKC activation or inhibition. This study demonstrates that activation of PKC decreases the excitability of spinal ventral neurons and that Vth can be modulated by multiple mechanisms.

Calcium-sensing receptor regulation of PTH-dependent calcium absorption by mouse cortical ascending limbs

Resting Ca2+ absorption by cortical thick ascending limbs (CALs) is passive and proceeds through the paracellular pathway. In contrast, parathyroid hormone (PTH) stimulates active, transcellular Ca2+ absorption ( J Ca). The Ca2+-sensing receptor (CaSR) is expressed on serosal membranes of CALs. In the present study, we tested the hypothesis that activation of the CAL CaSR indirectly inhibits passive Ca2+ transport and directly suppresses PTH-induced cellular J Ca. To test this theory, we measured J Ca and Na absorption ( J Na) by single perfused mouse CALs. Net absorption was measured microfluorimetrically in samples collected from tubules perfused and bathed in symmetrical HEPES-buffered solutions or those in which luminal Na+ was reduced from 150 to 50 mM. We first confirmed that Gd3+ activated the CaSR by measuring intracellular Ca2+ concentration ([Ca2+]i) in CALs loaded with fura 2. On stepwise addition of Gd3+ to the bath, [Ca2+]i increased, with a half-maximal rise at 30 μM Gd3+. J Ca and transepithelial voltage ( V e,) were measured in symmetrical Na+-containing solutions. PTH increased J Ca by 100%, and 30 μM Gd3+inhibited this effect. V e was unchanged by either PTH or Gd3+. Similarly, NPS R-467, an organic CaSR agonist, inhibited PTH-stimulated J Ca without altering V e. Neither PTH nor Gd3+affected J Na. Addition of bumetanide to the luminal perfusate abolished J Na and V e. These results show that CaSR activation directly inhibited PTH-induced transcellular J Caand that cellular Ca2+ and Na+ transport can be dissociated. To test the effect of CaSR activation on passive paracellular Ca2+ transport, J Ca was measured under asymmetrical Na conditions, in which passive Ca2+ transport dominates transepithelial absorption. PTH stimulated J Ca by 24% and was suppressed by Gd3+. In this setting, Gd3+ reduced V e by 32%, indicating that CaSR activation inhibited both transcellular and paracellular Ca2+transport. We conclude that the CaSR regulates both active transcellular and passive paracellular Ca2+ reabsorption but has no effect on J Na by CALs.

Effect of Bcl-2 on oxidant-induced cell death and intracellular Ca2+mobilization

The mechanism by which Bcl-2 inhibits cell death is unknown. It has been suggested that Bcl-2 functions as an antioxidant. Because Bcl-2 is localized mainly to the membranes of the endoplasmic reticulum (ER) and the mitochondria, which represent the main intracellular storage sites for Ca2+, we hypothesized that Bcl-2 might protect cells against oxidant injury by altering intracellular Ca2+homeostasis. To test this hypothesis, we examined the effect of oxidant treatment on viability in normal rat kidney (NRK) cells and in NRK cells stably transfected with Bcl-2 in the presence or absence of intracellular Ca2+, and we compared the effect of Bcl-2 expression on oxidant-induced intracellular Ca2+ mobilization and on ER and mitochondrial Ca2+pools. NRK cells transfected with Bcl-2 (NRK-Bcl-2) were significantly more resistant to H2O2-induced cytotoxicity than control cells. EGTA-AM, an intracellular Ca2+ chelator, as well as the absence of Ca2+ in the medium, reduced H2O2-induced cytotoxicity in both cell lines. Compared with controls, cells overexpressing Bcl-2 showed a delayed rise in intracellular Ca2+ concentration ([Ca2+]i) after H2O2treatment. After treatment with the Ca2+ ionophore ionomycin, Bcl-2-transfected cells showed a much quicker decrease after the maximal rise than control cells, suggesting stronger intracellular Ca2+ buffering, whereas treatment with thapsigargin, an inhibitor of the ER Ca2+-ATPases, transiently increased [Ca2+]iin control and in Bcl-2-transfected cells. Estimates of mitochondrial Ca2+ stores using an uncoupler of oxidative phosphorylation show that NRK-Bcl-2 cells have a higher capacity for mitochondrial Ca2+storage than control cells. In conclusion, Bcl-2 may prevent oxidant-induced cell death, in part, by increasing the capacity of mitochondria to store Ca2+.

Signaling the brain in systemic inflammation: which vagal branch is involved in fever genesis?

Recent evidence has suggested a role of abdominal vagal afferents in the pathogenesis of the febrile response. The abdominal vagus consists of five main branches (viz., the anterior and posterior celiac branches, anterior and posterior gastric branches, and hepatic branch). The branch responsible for transducing a pyrogenic signal from the periphery to the brain has not as yet been identified. In the present study, we address this issue by testing the febrile responsiveness of male Wistar rats subjected to one of four selective vagotomies: celiac (CBV), gastric (GBV), hepatic (HBV), or sham (SV). In the case of CBV, GBV, and HBV, only the particular vagal branch(es) was cut; for SV, all branches were left intact. After the postsurgical recovery (26–29 days), the rats had a catheter implanted into the jugular vein. On days 29–32, their colonic temperature (Tc) responses to a low dose (1 μg/kg) of Escherichia colilipopolysaccharide (LPS) were studied. Three days later, the animals were subjected to a 24-h food and water deprivation, and the effectiveness of the four vagotomies to induce gastric food retention, pancreatic hypertrophy, and impairment of the portorenal osmotic reflex was assessed by weighing the stomach and pancreas and measuring the specific gravity of bladder urine, respectively. Stomach mass, pancreas mass, and urine density successfully separated the four experimental groups into four distinct clusters, thus confirming that each type of vagotomy had a different effect on the indexes measured. The Tc responses of SV, CBV, and GBV rats to LPS did not differ and were characterized by a latency of ∼40 min and a maximal rise of 0.7 ± 0.1, 0.6 ± 0.1, and 0.9 ± 0.2°C, respectively. The fever response of the HBV rats was different; practically no Tc rise occurred (0.1 ± 0.2°C). The HBV appeared to be the only selective abdominal vagotomy affecting the febrile responsiveness. We conclude, therefore, that the hepatic vagus plays an important role in the transduction of a pyrogenic signal from the periphery to the brain.

In vivo stimulation of the beta(2)-adrenergic pathway increases expression of the Gi proteins and the alpha(2)A-adrenergic receptor genes in the pregnant rat myometrium

Cross-regulations between Gs and Gi mediated pathways controlling the adenylyl cyclase activity have been clearly demonstrated in vitro. To elucidate whether activation of the beta-adrenergic pathway in the pregnant myometrium might affect Gi proteins and alpha(2)-adrenergic receptors (ARs), we treated late pregnant rats from day 18 to day 21 with twice-daily administration of isoproterenol (8 mg/kg). This treatment increased myometrial cAMP levels and led after 76 h to a significant and maximal rise in the immunoreactive amount of myometrial Gi alpha 2 and Gi alpha 3 proteins (1.4- and 1.7-fold respectively) associated with a parallel increase of the steady-state levels of both Gi alpha 2 and Gi alpha 3 mRNA (1.6- and 1.9-fold respectively). Propranolol antagonized this response indicating the implication of the beta-adrenergic pathway. Nuclear run-on assays demonstrated that isoproterenol enhanced respectively by 1.3- and 1.2-fold the transcription rate of the Gi alpha 2 and Gi alpha 3 genes. Quantification of myometrial alpha(2)-ARs by [3H]rauwolscine binding revealed that the total number of receptors was also increased at 76 h by 1.7-fold when compared with controls, with no change in the affinity of the alpha(2)-ARs for the ligand. This effect was antagonized by propranolol. Quantification of both alpha(2A)- and alpha(2B)-subtypes by Northern blotting analysis demonstrated that this elevation was due to a selective increase of the alpha(2A)-subtype mRNAs. The present results indicate that in vivo stimulation of the beta-adrenergic pathway by isoproterenol increases both Gi alpha 2/Gi alpha 3 and alpha(2A)-AR expression in the pregnant rat myometrium. The possible contribution of such a mechanism in pregnancy-related changes of both entities is discussed.

Invasive hemodynamics and force-frequency relationships in open- versus closed-chest mice

We compared hemodynamics, ventricular function, and force-frequency relationships in six open-chest and six closed-chest anesthetized mice (FVB/N strain). Left ventricular (LV) pressure was measured with a 1.8- or 1.4-Fr Millar catheter placed via the right carotid artery and the LV apex in the closed- and open-chest state, respectively. Pacing was performed with electrodes placed either directly on atrial appendages (open chest) or with a 1-Fr bipolar catheter via the jugular vein (closed chest). Closed-chest animals had greater spontaneous heart rate (267 ± 106 vs. 147 ± 27 beats/min), LV systolic (81 ± 14 vs. 48 ± 9 mmHg) and diastolic pressures (11.2 ± 4.8 vs. 5.6 ± 2.4 mmHg), and maximal rise (+dP/d t max: 6,208 ± 2,519 vs. 3,682 ± 671 mmHg/s) and fall in pressure development (−dP/d t max: −6,094 ± 2,386 vs. −3,001 ± 399 mmHg/s). LV systolic pressure (98 ± 18 vs. 52 ± 11 mmHg), +dP/d t max (9,240 ± 2,459 vs. 5,777 ± 2,473 mmHg/s), and −dP/d t max(−8,375 ± 2,551 vs. −3,753 ± 1,170 mmHg/s) were significantly higher when animals were matched at a heart rate of 420 beats/min in closed-chest vs. open-chest animals. Biphasic force-frequency relationships were seen in all animals, but the critical heart rate was greater in the closed- than open-chest animals (432 ± 42 vs. 318 ± 42 beats/min). We conclude that 1) there are significant differences between invasive indexes of systolic and diastolic function between the closed- and open-chest preparations, 2) there is a biphasic force-frequency relationship in the anesthetized mouse, and 3) dP/d t max can be used to assess the cardiovascular phenotype.

Sequestration of glutamate-induced Ca2+ loads by mitochondria in cultured rat hippocampal neurons

1. Buffering of glutamate-induced Ca2+ loads in single rat hippocampal neurons grown in primary culture was studied with ratiometric fluorescent Ca2+ indicators. The hypothesis that mitochondria buffer the large Ca2+ loads elicited by glutamate was tested. 2. The relationship between glutamate concentration and the resulting increase in the free intracellular Ca2+ concentration ([Ca2+]i) reached an asymptote at 30 microM glutamate. This apparent ceiling was not a result of saturation of the Ca2+ indicator, because these results were obtained with the low-affinity (dissociation constant = 7 microM) Ca2+ indicator coumarin benzothiazole. 3. Five minutes of exposure to glutamate elicited concentration-dependent neuronal death detected 20-24 h later by the release of the cytosolic enzyme lactate dehydrogenase into the media. Maximal neurotoxicity was elicited at glutamate concentrations > or = 300 microM. The discrepancy between the glutamate concentration required to evoke a maximal rise in [Ca2+]i and the higher concentration necessary elicit maximal Ca(2+)-triggered cell death suggests that large neurotoxic Ca2+ loads are in part removed to a noncytoplasmic pool. 4. Treatment of hippocampal neurons with the protonophore carbonyl cyanide p-(trifluoro-methoxy) phenylhydrazone (FCCP; 1 microM, 5 min) greatly increased the amplitude of glutamate-induced [Ca2+]i transients, although it had little effect on basal [Ca2+]i. The effect of FCCP was more pronounced on responses elicited by stimuli that produced large Ca2+ loads. Similar results were obtained by inhibition of electron transport with antimycin A1. Neither agent, under the conditions described here, significantly depressed cellular ATP levels as indicated by luciferase-based ATP measurements, consistent with the robust anaerobic metabolism of cultured cells. Thus inhibition of mitochondrial function disrupted the buffering of glutamate-induced Ca2+ loads in a manner that was not related to changes in ATP. 5. Removal of extracellular Na+ for 20 min before exposure to N-methyl-D-aspartate (NMDA) (200 microM, 3 min), presumably reducing intracellular Na+, evoked a prolonged plateau phase in the recovery of the [Ca2+]i transient that resembled the mitochondrion-mediated [Ca2+]i plateau previously observed in sensory neurons. Return of extracellular Na+ immediately after exposure to NMDA increased the height and shortened the duration of the plateau phase. Thus manipulation of extracellular Na+ altered the plateau in a manner consistent with plateau height being modulated by intracellular Na+ levels. 6. In neurons depleted of Na+ and challenged with NMDA, a plateau resulted; during the plateau, application of FCCP in the absence of extracellular Ca2+ produced a large increase in [Ca2+]i. In contrast, similar treatment of cells that were not depleted of Na+ failed to increase [Ca2+]i. Thus Na+ depletion traps Ca2+ within an FCCP-sensitive intracellular store. 7. Glutamate-induced Ca2+ loads are sequestered by an intracellular store that had a low affinity and a high capacity for Ca2+, was released by FCCP, was sensitive to antimycin A1, and was modulated by intracellular Na+ levels. We conclude that mitochondria sequester glutamate-induced Ca2+ loads and suggest that Ca2+ entry into mitochondria may account for the poor correlation between glutamate-induced neurotoxicity and glutamate-induced changes in [Ca2+]i.