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.

IntroductionCardiovascular 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 MethodsHuman 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 Ki-values were calculated from dose–response curves at all temperatures, to evaluate temperature-dependent effects of both drugs.ResultsBoth drugs were able to reach the intracellular space at all hypothermic temperatures, with no reduction compared to normothermia. Sildenafil IC50 and Ki-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 Ki–values for inhibition of cellular cAMP and cGMP efflux.ConclusionSildenafil 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.

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.

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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-, β- 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] .