Moderate but not severe hypothermia increases intracellular cyclic AMP through preserved production and reduced elimination.

Rewarming from accidental hypothermia could be complicated by acute cardiac dysfunction but providing supportive pharmacotherapy at low core temperatures is challenging. Several pharmacological strategies aim to improve cardiovascular function by increasing cAMP in cardiomyocytes as well as cAMP and cGMP levels in vascular smooth muscle, but it is not clear what effects temperature has on cellular elimination of cAMP and cGMP. We therefore studied the effects of differential temperatures from normothermia to deep hypothermia (37 °C-20 °C) on cAMP levels in embryonic H9c2 cardiac cells and elimination of cAMP and cGMP by PDE-enzymes and ABC-transporter proteins. Our experiments showed significant elevation of intracellular cAMP in H9c2-cells at 30 °C but not 20 °C. Elimination of both cAMP and cGMP through ABC transport-proteins and PDE-enzymes showed a temperature dependent reduction. Accordingly, the increased cardiomyocyte cAMP-levels during moderate hypothermia appears an effect of preserved production and reduced elimination at 30 °C. This correlates with earlier in vivo findings of a positive inotropic effect of moderate hypothermia.

Pharmacodynamic properties for inhibition of cAMP- and cGMP elimination by pentoxifylline remain unaltered in vitro during hypothermia.

BACKGROUND: Rewarming from hypothermia is associated with severe complications, one of which is hypothermia-induced cardiac dysfunction. This condition is characterized by decreased cardiac output accompanied by increased total peripheral resistance. This contributes to mortality rate approaching 40%. Despite this, no pharmacological interventions are recommended for these patients below 30 °C. Raising the intracellular levels of cAMP and/or cGMP, through PDE3- and PDE5-inhibitors respectively, have showed the ability to alleviate hypothermia-induced cardiac dysfunction in vivo. Drugs that raise levels of both cAMP and cGMP could therefore prove beneficial in patients suffering from hypothermia-induced cardiac dysfunction. METHODS: The unselective PDE-inhibitor pentoxifylline was investigated to determine its ability to reach the intracellular space, inhibit PDE3 and PDE5 and inhibit cellular efflux of cAMP and cGMP at temperatures 37, 34, 30, 28, 24 and 20 °C. Recombinant human PDE-enzymes and human erythrocytes were used in the experiments. IC50-values were calculated at all temperatures to determine temperature-dependent changes. RESULTS: At 20 °C, the IC50-value for PDE5-mediated enzymatic breakdown of cGMP was significantly increased compared to normothermia (IC50: 39.4 µM ± 10.9 µM vs. 7.70 µM ± 0.265 µM, p-value = 0.011). No other significant changes in IC50-values were observed during hypothermia. CONCLUSIONS: This study shows that pentoxifylline has minimal temperature-dependent pharmacodynamic changes, and that it can inhibit elimination of both cAMP and cGMP at low temperatures. This can potentially be effective treatment of hypothermia-induced cardiac dysfunction. TRIAL REGISTRATION: Not applicable.

Treatment of Cardiovascular Dysfunction with PDE3-Inhibitors in Moderate and Severe Hypothermia-Effects on Cellular Elimination of Cyclic Adenosine Monophosphate and Cyclic Guanosine Monophosphate.

Introduction: Rewarming from accidental hypothermia is often complicated by hypothermia-induced cardiovascular dysfunction, which could lead to shock. Current guidelines do not recommend any pharmacological treatment at core temperatures below 30°C, due to lack of knowledge. However, previous in vivo studies have shown promising results when using phosphodiesterase 3 (PDE3) inhibitors, which possess the combined effects of supporting cardiac function and alleviating the peripheral vascular resistance through changes in cyclic nucleotide levels. This study therefore aims to investigate whether PDE3 inhibitors milrinone, amrinone, and levosimendan are able to modulate cyclic nucleotide regulation in hypothermic settings. Materials and methods: The effect of PDE3 inhibitors were studied by using recombinant phosphodiesterase enzymes and inverted erythrocyte membranes at six different temperatures-37°C, 34°C, 32°C, 28°C, 24°C, and 20°C- in order to evaluate the degree of enzymatic degradation, as well as measuring cellular efflux of both cAMP and cGMP. The resulting dose-response curves at every temperature were used to calculate IC50 and Ki values. Results: Milrinone IC50 and Ki values for cGMP efflux were significantly lower at 24°C (IC50: 8.62 ± 2.69 µM) and 20°C (IC50: 7.35 ± 3.51 µM), compared to 37°C (IC50: 22.84 ± 1.52 µM). There were no significant changes in IC50 and Ki values for enzymatic breakdown of cAMP and cGMP. Conclusion: Milrinone, amrinone and levosimendan, were all able to suppress enzymatic degradation and inhibit extrusion of cGMP and cAMP below 30°C. Our results show that these drugs have preserved effect on their target molecules during hypothermia, indicating that they could provide an important treatment option for hypothermia-induced cardiac dysfunction.

Treatment of Cardiovascular Dysfunction With PDE5-Inhibitors - Temperature Dependent Effects on Transport and Metabolism of cAMP and cGMP.

INTRODUCTION: Cardiovascular dysfunction is a potentially lethal complication of hypothermia. Due to a knowledge gap, pharmacological interventions are not recommended at core temperatures below 30°C. Yet, further cooling is induced in surgical procedures and survival of accidental hypothermia is reported after rewarming from below 15°C, advocating a need for evidence-based treatment guidelines. In vivo studies have proposed vasodilation and afterload reduction through arteriole smooth muscle cGMP-elevation as a favorable strategy to prevent cardiovascular dysfunction in hypothermia. Further development of treatment guidelines demand information about temperature-dependent changes in pharmacological effects of clinically relevant vasodilators. MATERIALS AND METHODS: Human phosphodiesterase-enzymes and inverted erythrocytes were utilized to evaluate how vasodilators sildenafil and vardenafil affected cellular efflux and enzymatic breakdown of cAMP and cGMP, at 37°C, 34°C, 32°C, 28°C, 24°C, and 20°C. The ability of both drugs to reach their cytosolic site of action was assessed at the same temperatures. IC50- and K i -values were calculated from dose-response curves at all temperatures, to evaluate temperature-dependent effects of both drugs. RESULTS: Both drugs were able to reach the intracellular space at all hypothermic temperatures, with no reduction compared to normothermia. Sildenafil IC50 and K i -values increased during hypothermia for enzymatic breakdown of both cAMP (IC50: 122 ± 18.9 µM at 37°C vs. 269 ± 14.7 µM at 20°C, p < 0.05) and cGMP (IC50: 0.009 ± 0.000 µM at 37°C vs. 0.024 ± 0.004 µM at 32°C, p < 0.05), while no significant changes were detected for vardenafil. Neither of the drugs showed significant hypothermia-induced changes in IC50 and K i- values for inhibition of cellular cAMP and cGMP efflux. CONCLUSION: Sildenafil and particularly vardenafil were ableto inhibit elimination of cGMP down to 20°C. As the cellular effects of these drugs can cause afterload reduction, they show potential in treating cardiovascular dysfunction during hypothermia. As in normothermia, both drugs showed higher selectivity for inhibition of cGMP-elimination than cAMP-elimination at low core temperatures, indicating that risk for cardiotoxic side effects is not increased by hypothermia.

Inhibition of ABCC5-mediated cGMP transport by progesterone, testosterone and their analogues.

The biodynamics and biokinetics of sex hormones are complex. In addition to the classical steroid receptors (nuclear receptors), these hormones act through several non-genomic mechanisms. Modulation of ABC-transporters by progesterone represents a non-genomic mechanism. In the present study, we employed inside out vesicles from human erythrocytes to characterize high affinity cGMP transport by ABCC5 (member 5 of the ATP-Binding Cassette subfamily C). Progesterone and testosterone inhibited the transport with respective Ki of 1.2 ± 0.3 and 2.0 ± 0.6 µmol/L. We used virtual ligand screening (VLS) to identify analogues to progesterone and testosterone. A large number of substances were screened in silico and the 19 most promising candidates were screened in vitro. Each substance was tested for a concentration of 10 µmol/L. The range of cGMP transport reduction was 21.5% to 86.2% for progesterone analogues and 8.6% to 93.8 % for testosterone analogues. Three of the most potent test compounds (TC) of each analogue class, in addition to progesterone and testosterone, were characterized for concentrations from 1 nanomol/L to 1 mmol/L. The progesterone analogues showed following Ki-values (µmol/L): TC-08: 0.61, TC-16: 0.66 and TC-15: 9.3. The Ki-values (µmol/L) for the testosterone analogues were: TC-18: 0.10, TC-07: 0.67 andTC-05: 2.0. The present study shows that VLS may be a versatile tool in the development of membrane transport modulating agents (MTMAs).

Transfer of triptans into human breast milk and estimation of infant drug exposure through breastfeeding.

Clinical data on the transfer of triptans into human breast milk remain scarce. In a lactation study including 19 breastfeeding women with migraine, we examined the excretion of six different triptans into milk. Following intake of a single dose, each participant collected seven breast milk samples at predefined intervals up to 24 hours after dose. Triptan concentrations in milk were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Infant drug exposure was estimated by calculating the relative infant dose (RID). Twenty-two breast milk sample sets were obtained for sumatriptan (n = 8), rizatriptan (n = 5), zolmitriptan (n = 4), eletriptan (n = 3), almotriptan (n = 1) and naratriptan (n = 1). Based on the average concentration in milk throughout the day, estimated mean RIDs (with range in parenthesis) were as follows: eletriptan 0.6% (0.3%-0.8%), sumatriptan 0.7% (0.2%-1.8%), rizatriptan 0.9% (0.3%-1.4%), almotriptan 1.8% (-), zolmitriptan 2.1% (0.7%-5.3%) and naratriptan 5.0% (-). Infant drug exposure through breastfeeding appears to be low and indicates that use of the triptans in this study is compatible with breastfeeding. Naratriptan may not be first choice in breastfeeding mothers initiating triptans during the neonatal period.

Molecular modeling study of the testosterone metabolizing enzyme UDP-glucuronosyltransferase 2B17.

The dominant sex hormone testosterone is mainly metabolized by liver enzymes belonging to the uridine-diphospho (UDP) glucuronosyltransferase (UGT) family. These enzymes are the main phase II enzymes, and they have an important role in the detoxification of endogenous and exogenous compounds in humans. The aim of the present study was to improve the understanding of the binding properties of UGT2B17. A homology modelling procedure was used to generate models of the UGT2B17 enzyme based on templates with known crystal structures. Molecular docking of inhibitors was performed to gain further insights in the interactions between ligand and binding site, and to determine which of the models had the best accuracy. ROC curves were made to evaluate the ability of the models to differentiate between binders (inhibitors) and non-binders (decoys). When comparing the four models, which were based on four different crystal structures, the model based on the 4AMG crystal structure was the most accurate in distinguishing between true binders and non-binders. Investigating pharmacological UGT2B17 inhibition may provide novel treatment for patients with low testosterone levels. Such treatment may elevate endogenous testosterone levels and provide a more predictable increase in serum concentrations rather than un-physiological elevation of serum levels through direct treatment with testosterone, and this could be favorable both for giving a predictable treatment regime with reduced chances of serious adverse effects. The present study may serve as a tool in the search for novel drugs aiming for increasing testosterone levels.

Data on RT-qPCR assay of nuclear progesterone receptors (nPR), membrane progesterone receptors (mPR) and progesterone receptor membrane components (PGRMC) from human uterine endometrial tissue and cancer cells of the Uterine Cervix.

A previous investigation showed that the endometrium normalized in women with endometrial hyperplasia after three months treatment with high dose levonorgestrel IUS (intrauterine system) [1] . The effect was maintained even if immunohistochemical analyses of the endometrium showed that nuclear progesterone receptors (nPRs) were completely downregulated. These observations indicated that some type of non-genomic effect existed [2]. We conducted new investigations of endometrial hyperplasia, now with 6 months low dose levonorgestrel IUS treatment. Again, the growth disturbances were reversed with normalization of the endometrium [3,4]. In the context of these studies, RT-qPCR analyses of the endometrium were performed before and after treatment, to determine expression of nuclear progesterone receptors (nPRA+B and nPRB), membrane progesterone receptors (mPR, α-, ß- and γ-subtypes) and progesterone receptor membrane components (PGRMC1and PGRMC2). The human cervical cell line (C-4 I) [5] with no detectable nPRs [6,7] , was included in the investigation as biological control .The gene expression of nPRs, mPRs and PGRMCs was determined in the logarithmic growth phase. Tissue and cellular mRNA was determined with RT-qPCR and used as a surrogate marker for receptor (protein) expression. The present data are connected to the related article entitled "Expression of nuclear progesterone receptors (nPRs), membrane progesterone receptors (mPRs) and progesterone receptor membrane components (PGRMCs) in the human endometrium after 6 months levonorgestrel low dose intrauterine therapy" [8].

Expression of nuclear progesterone receptors (nPRs), membrane progesterone receptors (mPRs) and progesterone receptor membrane components (PGRMCs) in the human endometrium after 6 months levonorgestrel low dose intrauterine therapy.

The classical steroid receptors (nuclear receptors), including those for progesterone (nPRs), are thoroughly characterized. The knowledge about so-called non-genomic effects, which are mediated by extra-nuclear initiated signals, has increased immensely the last decades. In a previous clinical study of endometrial hyperplasia, we observed that the antiproliferative progestin effect persisted after 3 months treatment with levonorgestrel (LNG) intrauterine system (IUS) even with a complete downregulation of nPRs. This raised the question of what other mechanisms than signaling through nPRs could explain such an observation. In the present study, RT-qPCR was employed to characterize mRNA expression for nPRs, membrane progesterone receptors (mPRs) and progesterone receptor membrane components (PGRMCs) in women (n = 42) with endometrial hyperplasia that received intrauterine low dose LNG for 6 months. At the end of this period endometrial tissue showed that nPRs were virtually completely downregulated (≈ 10 % of baseline) whereas the levels of remaining mPRs, subtype-α, -ß and -γ were 76 %, 59 % and 73 % of baseline, respectively. PGRMC1 was downregulated to 15 % of baseline, in contrast to PGRMC2, which was upregulated to about 30 % above baseline. We used human cancer cells from uterine cervix (C-4I cells) as control. Progesterone caused a concentration-dependent antiproliferative effect but in several and separate studies, we were unable to detect nPRs (immunocytochemistry) in the C-4I cells. The use of RT-qPCR showed that nPRs were undetectable in C-4I cells, in contrast to mPRs and PGRMCs with a distinct mRNA expression. The present study suggests that mPRs and/or PGRMCs preserve the antiproliferative effect of LNG in the human endometrium and are responsible for the concentration-dependent antiproliferative effect of progesterone in C-4I cells.

Identification and experimental confirmation of novel cGMP efflux inhibitors by virtual ligand screening of vardenafil-analogues.

BACKGROUND: Clinical studies have reported overexpression of PDE5 and elevation of intracellular cyclic GMP in various types of cancer cells. ABCC5 transports cGMP out of the cells with high affinity. PDE5 inhibitors prevent both cellular metabolism and cGMP efflux by inhibiting ABCC5 as well as PDE5. Increasing intracellular cGMP is hypothesized to promote apoptosis and growth restriction in tumor cells and also has potential for clinical use in treatment of cardiovascular disease and erectile dysfunction. Vardenafil is a potent inhibitor of both PDE5 and ABCC5-mediated cGMP cellular efflux. Nineteen novel vardenafil analogs that have been predicted as potent inhibitors by VLS were chosen for tests of their ability to inhibit ATP- dependent transport of cGMP by measuring the accumulation of cyclic GMP in inside-out vesicles. AIM: In this study, we investigated the ability of nineteen new compounds to inhibit ABCC5- mediated cGMP transport. We also determined the Ki values of the six most potent compounds. METHODS: Preparation of human erythrocyte inside out vesicles and transport assay. RESULTS: Ki values for six of nineteen compounds that showed more than 50 % inhibition of cGMP transport in the screening test were determined and ranged from 1.1 to 23.1 µM. One compound was significantly more potent than the positive control, sildenafil. CONCLUSION: Our findings show that computational screening correctly identified vardenafil-analogues that potently inhibit cGMP efflux-pumps from cytosol and could have substantial clinical potential in treatment of patients with diverse disorders.

The role of OAT2 (SLC22A7) in the cyclic nucleotide biokinetics of human erythrocytes.

The present study was conducted to characterise the transporter(s) responsible for the uptake of cyclic nucleotides to human erythrocytes. Western blotting showed that hRBC expressed OAT2 (SLC22A7), but detection of OAT1 (SLC22A6), or OAT3 (SLC22A8) was not possible. Intact hRBC were employed to clarify the simultaneous cyclic nucleotide egression and uptake. Both these opposing processes were studied. The Km -values for high affinity efflux was 3.5 ± 0.1 and 39.4 ± 5.7 µM for cGMP and cAMP, respectively. The respective values for low affinity efflux were 212 ± 11 and 339 ± 42 µM. The uptake was characterised with apparently low affinity and similar Km -values for cGMP (2.2 mM) and cAMP (0.89 mM). Using an iterative approach in order to balance uptake with efflux, the predicted real Km -values for uptake were 100-200 µM for cGMP and 50-150 µM for cAMP. The established OAT2-substrate indomethacin showed a competitive interaction with cyclic nucleotide uptake. Creatinine, also an OAT2 substrate, showed saturable uptake with a Km of 854 ± 98 µM. Unexpectedly, co-incubation with cyclic nucleotides showed an uncompetitive inhibition. The observed Km -values were 399 ± 44 and 259 ± 30 µM for creatinine, in the presence of cGMP and cAMP, respectively. Finally, the OAT1-substrate para-aminohippurate (PAH) showed some uptake (Km -value of 2.0 ± 0.4 mM) but did not interact with cyclic nucleotide or indomethacin transport.

Inhibition of PDE5A1 guanosine cyclic monophosphate (cGMP) hydrolysing activity by sildenafil analogues that inhibit cellular cGMP efflux.

OBJECTIVES: To determine the ability of 11 sildenafil analogues to discriminate between cyclic nucleotide phosphodiesterases (cnPDEs) and to characterise their inhibitory potencies (Ki values) of PDE5A1-dependent guanosine cyclic monophosphate (cGMP) hydrolysis. METHODS: Sildenafil analogues were identified by virtual ligand screening (VLS) and screened for their ability to inhibit adenosine cyclic monophosphate (cAMP) hydrolysis by PDE1A1, PDE1B1, PDE2A1, PDE3A, PDE10A1 and PDE10A2, and cGMP hydrolysis by PDE5A, PDE6C, PDE9A2 for a low (1 nm) and high concentration (10 µm). Complete IC50 plots for all analogues were performed for PDE5A-dependent cGMP hydrolysis. Docking studies and scoring were made using the ICM molecular modelling software. KEY FINDINGS: The analogues in a low concentration showed no or low inhibition of PDE1A1, PDE1B1, PDE2A1, PDE3A, PDE10A1 and PDE10A2. In contrast, PDE5A and PDE6C were markedly inhibited to a similar extent by the analogues in a low concentration, whereas PDE9A2 was much less inhibited. The analogues showed a relative narrow range of Ki values for PDE5A inhibition (1.2-14 nm). The sildenafil molecule was docked in the structure of PDE5A1 co-crystallised with sildenafil. All the analogues had similar binding poses as sildenafil. CONCLUSIONS: Sildenafil analogues that inhibit cellular cGMP efflux are potent inhibitors of PDE5A and PDE6C.

Altered pharmacological effects of adrenergic agonists during hypothermia.

Rewarming from accidental hypothermia is often complicated by hypothermia-induced cardiac dysfunction, calling for immediate pharmacologic intervention. Studies show that although cardiac pharmacologic support is applied when rewarming these patients, a lack of updated treatment recommendations exist. Mainly due to lack of clinical and experimental data, neither of the international guidelines includes information about pharmacologic cardiac support at temperatures below 30 °C. However, core temperature of accidental hypothermia patients is often reduced below 30 °C. Few human studies exploring effects of adrenergic drugs during hypothermia have been published, and therefore prevailing information is collected from pre-clinical studies. The most prominent finding in these studies is an apparent depressive effect of adrenaline on cardiac function when used in doses which elevate cardiac output during normothermia. Also noradrenaline and isoprenaline largely lacked positive cardiac effects during hypothermia, while dopamine is a more promising drug for supporting cardiac function during rewarming. Data and information from these studies are in support of the prevailing notion; not to use adrenergic drugs at core temperatures below 30 °C.

Negative inotropic effects of epinephrine in the presence of increased ß-adrenoceptor sensitivity during hypothermia in a rat model.

BACKGROUND: Animal studies show reduced inotropic effects of cardiac ß-adrenoceptor agonists like epinephrine (Epi) during hypothermia and rewarming, while drugs targeting other pharmacological mechanisms have positive effects. This study therefore aimed to determine ß-adrenoceptor sensitivity in isolated cardiomyocytes and investigate hemodynamic effects of Epi and its ability to stimulate cardiac ß-adrenoceptors at different temperatures in vivo. METHODS: Isolated rat myocardial cells were incubated with the radioactive ß-adrenoceptor ligand [(3)H]-CGP12177 and propranolol, used as a displacer. Cells were subjected to normothermia (37 °C) or hypothermia (15 °C). After incubation, radioactivity was measured to estimate ß-adrenoceptor affinity for propranolol (IC50), as a measure of ß-adrenoceptor sensitivity. In separate in vivo experiments, Epi (1.25 µg/min) was administered the last 5min of experiments in normothermic (37 °C, 5h), hypothermic (4h at 15 °C) and rewarmed rats (4h at 15 °C, and subsequently rewarmed to 37 °C). Hemodynamic parameters were monitored during infusion. Hearts were thereafter freeze-clamped and tissue cAMP was measured. RESULTS: In vitro measurements of IC50 for propranolol showed a hypothermia-induced increase in ß-adrenoceptor sensitivity at 15 °C. Corresponding in vivo experiments at 15 °C showed decreased cardiac output and stroke volume, whereas total peripheral resistance (TPR) increased during Epi infusion, simultaneous with a 4-fold cAMP increase. CONCLUSIONS: This experiment shows a hypothermia-induced in vivo and in vitro increase of cardiac ß-adrenoceptor sensitivity, and simultaneous lack of inotropic effects of Epi in the presence of increased TPR. Our findings therefore indicate that hypothermia-induced reduction in inotropic effects of Epi is due to substantial elevation of TPR, rather than ß-adrenoceptor dysfunction.

Modulation of high affinity ATP-dependent cyclic nucleotide transporters by specific and non-specific cyclic nucleotide phosphodiesterase inhibitors.

Intracellular cyclic nucleotides are eliminated by phosphodiesterases (PDEs) and by ATP Binding cassette transporters such as ABCC4 and ABCC5. PDE5 and ABCC5 have similar affinity for cGMP whereas ABCC5 has much higher affinity for cGMP compared with cAMP. Since the substrate (cGMP) is identical for these two eliminatory processes it is conceivable that various PDE inhibitors also modulate ABCC5-transport. Cyclic GMP is also transported by ABBC4 but the affinity is much lower with a Km 50-100 times higher than for that of ABBCC5. The present study aimed to determine Ki-values for specific or relative specific PDE5 inhibitors (vardenafil, tadalafil, zaprinast and dipyridamole) and the non-specific PDE inhibitors (IBMX, caffeine and theophylline) for ABCC5 and ABCC4 transport. The transport of [(3)H]-cGMP (2 µM) was concentration-dependently inhibited with the following Ki-values: vardenafil (0.62 µM), tadalafil (14.1 µM), zaprinast (0.68 µM) and dipyridamole (1.2 µM), IBMX (10 µM), caffeine (48 µM) and theophylline (69 µM). The Ki-values for the inhibition of the [(3)H]-cAMP (2 µM) transport were: vardenafil (3.4 µM), tadalafil (194 µM), zaprinast (2.8 µM), dipyridamole (5.5 µM), IBMX (16 µM), caffeine (41 µM) and theophylline (85 µM). The specificity for ABCC5 we defined as ratio between Ki-values for inhibition of [(3)H]-cGMP and [(3)H]-cAMP transport. Tadalafil showed the highest specificity (Ki-ratio: 0.073) and caffeine the lowest (Ki-ratio: 1.2).

Increased gene expression of the ABCC5 transporter without distinct changes in the expression of PDE5 in human cervical cancer cells during growth.

Carcinoma of the uterine cervix represents the second most frequent female malignancy worldwide, but few biochemical tumour markers have been implemented into clinical practice. Elevated extracellular guanosine 3', 5'-cyclic monophosphate (cGMP) levels have been reported to be a sensitive, early and reliable marker for screening relapse in carcinoma of the uterine cervix. The mechanism behind this observation remains unknown. The possibility exists that the cancer cells develop resistance to the antiproliferative effect of high intracellular cGMP levels. The enhanced cGMP expression may originate from either an increase in cellular export capacity by increased expression of member 5 in subfamily C of ATP-Binding-Cassette transporters (ABCC5), or increased substrate (cGMP) levels for this pump. The latter situation occurs with increased expression of inducible nitric oxide synthase (iNOS) and/or soluble guanylyl cyclase (sGC) and/or reduced expression of member 5 of the cyclic nucleotide phosphodiesterases (PDE5). Four transformed human cell lines derived from carcinomas of the uterine cervix (C-4 I, C-33 A, SiHa and ME-180 cells) and one non-transformed human cell line (WI-38) were included in the study in order to unveil which biokinetic components are involved. The expressions of iNOS, sGC, PDE5 and ABCC5 in the initial and final phase of the exponential growth curve were compared. Assuming that the WI-38 control cells mimic the situation in a normal tissue, iNOS remains un-expressed during proliferation, and the expression of sGC is low but shows a clear increase during exponential growth. PDE5 is highly expressed and increases (≈130%) during growth whereas ABCC5 exhibited low to moderate expression, with a moderate increase (≈40%) during growth. The malignant cells exhibited moderate ABCC5 expression with a distinct increase during exponential growth, whereas PDE5 expression remained virtually unchanged. Dysregulation of the cGMP biokinetics in growing malignant cells may account for the elevation of extracellular cGMP observed in patients with carcinoma of the uterine cervix.

Novel cGMP efflux inhibitors identified by virtual ligand screening (VLS) and confirmed by experimental studies.

Elevated intracellular levels of cyclic guanosine monophosphate (cGMP) may induce apoptosis, and at least some cancer cells seem to escape this effect by increased efflux of cGMP, as clinical studies have shown that extracellular cGMP levels are elevated in various types of cancer. The human ATP binding cassette (ABC) transporter ABCC5 transports cGMP out of cells, and inhibition of ABCC5 may have cytotoxic effects. Sildenafil inhibits cGMP efflux by binding to ABCC5, and in order to search for potential novel ABCC5 inhibitors, we have identified sildenafil derivates using structural and computational guidance and tested them for the cGMP efflux effect. Eleven compounds from virtual ligand screening (VLS) were tested in vitro, using inside-out vesicles (IOV), for inhibition of cGMP efflux. Seven of 11 compounds predicted by VLS to bind to ABCC5 were more potent than sildenafil, and the two most potent showed K(i) of 50-100 nM.

Changes in cardiovascular effects of dopamine in response to graded hypothermia in vivo.

OBJECTIVE: Inotropic drugs are frequently administered in hypothermic patients to support an assumed inadequate circulation, but their pharmacologic properties at reduced temperatures are largely unknown. Thus we estimated dopamine pharmacokinetics as well as left ventricular function and global hemodynamics after dopamine infusions at various core temperatures in a pig model of surface cooling and rewarming. DESIGN: Prospective, randomized, open, placebo-controlled experimental study. SETTING: University-affiliated animal research laboratory. SUBJECTS: Sixteen healthy, anesthetized juvenile (2-3 months) castrated male pigs. INTERVENTIONS: After normothermic infusions of dopamine at different doses (4, 8, and 16 µg/kg/min), effects of dopamine (n = 8) or saline (n = 8) were tested at 25 °C and during rewarming (30-34 °C). MEASUREMENTS AND MAIN RESULTS: Dopamine half-time was 5.4 ± 0.7 min at normothermia, increased to 11.6 ± 0.8 min at 25 °C, but returned to control during rewarming at 34-35 °C. Dopamine infusion at 25 °C elevated dopamine plasma concentration four-fold compared to the same infusion rate at normothermia, leading to increased systemic vascular resistance index not seen at normothermia. Also, in contrast to the dopamine-mediated increase in cardiac index observed at normothermia, high-dose dopamine at 25 °C left cardiac index unchanged despite a concomitant increase in heart rate, since stroke index decreased by 43%. During rewarming, cardiovascular effects of dopamine at moderate hypothermia (30-34 °C) were principally similar to responses during normothermia. CONCLUSIONS: Pharmacodynamic effects and pharmacokinetics of dopamine are maintained during the rewarming phase at moderate hypothermia. However, at 25 °C dopamine pharmacokinetics were seriously altered and dopamine failed to increase cardiac index since stroke index was reduced with incrementing dosages. Properties of the low-flow, high-viscosity circulatory state, combined with altered pharmacokinetics of dopamine, may explain lack of beneficial--and potentially harmful--effects from dopamine administration at 25 °C.