Inhibitors of cytochrome P-450 reduce cyclic GMP stimulation by glyceryl trinitrate in LLC-PK1 kidney epithelial cells

Summary The inhibitor of cytochrome P-450 cimetidine was used to asses the role of cytochrome P-450-dependent enzymes for cyclic GMP stimulation by glyceryl trinitrate in a kidney epithelial cell line (LLC-PK₁). Pretreatment of the cells with 0.1 mmol/1 cimetidine markedly decreased cyclic GMP stimulation by glyceryl trinitrate (0.03 –1 mol/l). In the presence of 0.1 mmol/1 cimetidine, the 14-fold cyclic GMP stimulation observed at 1 mol/l glyceryl trinitrate was reduced by 66%. Glyceryl trinitrate-induced cyclic GMP stimulation remained unaltered by ranitidine (0.1 mmol/1), which has a much lower affinity for the cytochrome P-450 enzyme system. Another inhibitor of cytochrome P-450, miconazole (0.1 mmol/1), also attenuated glyceryl trinitrate-induced cyclic GMP stimulation. In contrast, cimetidine and miconazole did not affect cyclic GMP stimulation by sodium nitroprusside that spontaneously releases nitric oxide. These results suggest that in intact cells, glyceryl trinitrate-induced cyclic GMP stimulation is dependent on cytochrome P-450 enzymes which may be relevant for nitric oxide formation from organic nitrates.Send offprint requests to K. Schrör at the above address     

Cyclic GMP stimulation by vasopressin in LLC-PK1 kidney epithelial cells is l-arginine-dependent

Summary The l-arginine antagonist N^G-monomethyl-l-arginine has been shown to inhibit nitric oxide formation from l-arginine in endothelial cells. In the present study N^G-monomethyl-l-arginine was used to assess the role of l-arginine for cyclic GMP stimulation by vasopressin in a kidney epithelial cell line (LLC-PK₁). Preincubation of cells with 1 mol/l, 10 mol/l and 100 mol/l N^G-monomethyl-l-arginine decreased cyclic GMP stimulation at 1 mol/l vasopressin by 25%, 71% and 90%, respectively. This inhibition by N^G-monomethyl-l-arginine was markedly reduced by l-arginine (2 mmol/1) but not d-arginine (2 mmol/1). Cyclic GMP stimulation by the calcium ionophore A23187 was also inhibited by N^G-monomethyl-l-arginine and enantioselectively restored by l-arginine. However, N^G-monomethyl-l-arginine did not affect cyclic GMP stimulation by sodium nitroprusside that spontaneously releases nitric oxide. These results suggest that, in kidney epithelial cells, vasopressin induces nitric oxide formation from l-arginine leading to activation of soluble guanylate cyclase. It is concluded that nitric oxide formation from l-arginine is not only responsible for endothelium-dependent relaxation but may be a more general pathway with regulatory function for intracellular guanylate cyclase activity.Send offprint requests to K. Schror at the above address     

The effect of nitric oxide-donating vasodilators on monocyte chemotaxis and intracellular cGMP concentrations in vitro

Summary The effects of sodium nitroprusside (SNP) and 3-morpholino sydnonimine (SIN-1), isosorbide dinitrate (ISDN) and glyceryl trinitrate (GTN), and molsidomine (the inactive precursur of SIN-1) on monocyte chemotaxis and cyclic GMP (cGMP) concentration were studied.SNP and SIN-1 inhibited monocyte N-formyl-methionyl-leucyl-phenylalanine-stimulated migration and increased cGMP concenrations in a dose-dependent (>10^–5 mol·l^–1) and time-dependent manner. Furthermore, 8-bromo cGMP inhibited monocyte chemotaxis in a dose-dependent fashion. In contrast, ISDN, GTN and molsidomine did not alter monocyte migration or cGMP concentration.These results support earlier observations that nitric oxide inhibits monocyte function in vitro via a cGMP-mediated mechanism.The differential effects of the spontaneous and thiol-dependent NO-donating nitrovasodilators on monocyte function suggests that monocytes, like platelets, are not able to directly metabolise ISDN and GTN. If similar observations can be made in vivo, it is possible that certain nitrovasodilators might be used therapeutically to inhibit monocyte function, for example during atherogenesis.     

Reduced nitric oxide release causes nitrate tolerance in the intact coronary circulation.

We investigated the possible involvement of reduced nitric oxide (NO) formation in development of nitrate tolerance in an intact organ circulation. NO formation was measured spectrophotometrically on-line in the coronary effluent of Langendorff hearts of rabbits. Short-term (3 min) infusion of glyceryl trinitrate (GTN, 40 microM) or a sydnonimine (SIN-1, 2.3 microM), the active metabolite of molsidomine, into the coronary inflow tract resulted in a decrease in coronary vascular resistance and NO release into the coronary effluent. Pretreatment with 250 microM GTN for 30 min resulted in considerably reduced NO formation and coronary vasodilation, whereas NO release and coronary vasodilation subsequent to SIN-1 remained unchanged. In hearts pretreated with 250 microM SIN-1 for 30 min, there was no effect on GTN- or SIN-1-induced vasodilation and NO release. Studies of cyclic GMP formation in rat lung fibroblasts further indicated that GTN bioconversion rather than desensitization of the soluble guanylate cyclase is involved in GTN tolerance. These data suggest metabolic, endothelium-independent NO release from GTN during passage through the coronary circulation. This NO release is reduced in nitrate-tolerant cells and appears to be the major cause of nitrate tolerance in intact circulatory systems.     

Endothelium- and sydnonimine-induced responses of native and cultured aortic smooth muscle cells are not impaired by nitroglycerin tolerance

Summary Tolerance to the cyclic GMP-mediated vasodilator action of nitroglycerin develops with prolonged exposure and may be mediated either by formation of less nitric oxide from nitroglycerin or by desensitization of soluble guanylate cyclase to activation with nitric oxide. In the latter case, smooth muscle cells tolerant to nitroglycerin should show cross-tolerance to nitric oxide released from sydnonimines and endothelial cells (endothelium-derived relaxing factor).Therefore cultured smooth muscle cells from rabbit aorta were pretreated for 1 h with vehicle or high concentrations (0.55 mM) of nitroglycerin or the sydnonimine SIN-1. The formation of cyclic GMP induced by subsequent small doses of nitroglycerin, sydnonimine SIN-1 and endothelium-derived relaxing factor (released from cultured endothelial cells) was compared with the changes in activation of soluble guanylate cyclase, cyclic GMP formation and vasodilation in response to the same stimuli in similarly pretreated segments from rabbit thoracic aortae.Both cultured and native smooth muscle cells remained responsive to stimulation with sydnonimine SIN-1 and endothelium-derived relaxing factor after pretreatment with nitroglycerin, vehicle, or sydnonimine SIN-1, even though they were tolerant to nitroglycerin after pretreatment with nitroglycerin. In contrast, activation of soluble guanylate cyclase by nitroglycerin and sydnonimine SIN-1 was attenuated in homogenates of nitrate-tolerant aortae. The findings suggest that nitroglycerin tolerance in intact cells does not involve desensitization of soluble guanylate cyclase, because in intact cells nitrate tolerance can be overcome by direct activators of soluble guanylate cyclase. Send offprint requests to A. Mülsch at the above address     

Biphasic relaxant curves to glyceryl trinitrate in rat aortic rings

Summary The concentration-effect curve for the relaxant effects of glyceryl trinitrate (GTN) in rat aortic rings consisted of two phases with IC₅₀ values of 0.1 M for Phase I and 14 M for Phase II. Incubation of tissues with oxyhaemoglobin or the induction of tolerance to GTN abolished responses occurring in Phase I but were without effect on Phase II relaxant responses. Both phases of the relaxant curve appeared to involve cyclic GMP since responses were (i) potentiated by the cyclic GMP phosphodiesterase inhibitor zaprinast (M & B 22948) and (ii) inhibited by methylene blue and LY83583, agents which inhibit soluble guanylate cyclase. The latter agents inhibited Phase I responses in a non-surmountable manner while Phase II responses were shifted to the right without effect on the maximal response. Neither phase of relaxation involved stimulation of the Na⁺/K⁺ ATPase pump since treatment of tissues with ouabain or K⁺-free solutions did not alter the GTN biphasic curve. Phase I relaxant responses to GTN resembled those to the endothelium-dependent relaxant acetylcholine, since oxyhaemoglobin and methylene blue were non-surmountable antagonists; however there was no cross tolerance to acetylcholine in GTN tolerant tissues. Phase II relaxant responses resembled those obtained with sodium nitroprusside (SNP) since neither oxyhaemoglobin nor the induction of tolerance to GTN altered the response to SNP. These results indicate that there are two distinct mechanisms of relaxation for GTN in rat aortic rings; however both mechanisms appear to involve cyclic GMP as the second messenger. Send offprint requests to E. Malta at the above address     

Exogenous Modification of Nitrovasodilator-Induced Cyclic GMP Formation in Human Lymphocytes

Abstract: The effects of exogenous guanosine 5′-triphosphate (GTP), guanosine 5′-(γy-thio)triphosphate (GTPγS), cysteine and Trolox C., a water soluble vitamin E analogue, were studied on basal and nitrovasodilator-induced cyclic GMP formation in isolated human lymphocytes. Incubation of lymphocytes in the presence of GTP (0.1 mM) and GTPγS (0.1 mM) increased cyclic GMP more than twofold. SIN-1 and sodium nitroprusside dose-dependently increased cyclic GMP, but nitroglycerin and sodium nitrite were ineffective. GTP and GTPγS potentiated SIN-1- and sodium nitroprusside-induced cyclic GMP formation. In the presence of GTP and GTPγS, nitroglycerin, but not sodium nitrite, was able to increase lymphocyte cyclic GMP. Cysteine (1 mM) enhanced cyclic GMP formation induced by sodium nitroprusside and nitroglycerin. Trolox C (0.1 mM) potentiated SIN-1-induced cyclic GMP formation. These results indicate that exogenous GTP and GTPγS enhance guanylate cyclase stimulation by spontaneous nitric oxide releasers and nitroglycerin in lymphocytes. Cysteine, a redox-compound and Trolox C, an antioxidant, have different effects on guanylate cyclase activation by nitric oxide releasers, SIN-1 and sodium nitroprusside.     

On-line measurement of nitric oxide release from organic nitrates in the intact coronary circulation

Summary This study determines the release of nitric oxide (NO) from the coronary circulation of Langendorff hearts of rabbits, subsequent to administration of glyceryl trinitrate (GTN) and SIN-1. NO was measured on-line in the coronary effluent by the oxyhaemoglobin technique. Infusion of either GTN (10–40 moles/l) or SIN-1 (0.1–2.3 moles/l) into the coronary inflow resulted in a concentration-dependent NO release into the coronary effluent and a decrease in the coronary vascular resistance. NO generation from SIN-1 was identical with and without passage of the coronary circulation whereas NO generation from GTN was only detected after passage of the coronary vascular bed. NO generation by both substances was in the same range as endogenous NO release by two endothelium-dependent vasodilators, bradykinin (0.05 moles/l) and substance P (0.05 moles/l). Oxyhaemoglobin used for the assay of NO, inhibited the relaxation by SIN-1, but did not reduce vessel relaxations induced by GTN or iloprost, a stable prostacyclin analogue. Removal of the coronary endothelium by trypsin or pretreatment with L-N^G-Monomethyl-arginine (30 moles/l) did neither affect NO release from GTN and SIN-1 nor the vasodilatory effect of both substances. These data are the first to directly demonstrate endothelium-independent NO release from organic nitrates during passage of an intact organ circulation. They additionally suggest a subendothelial site of metabolic NO formation from GTN. Send offprint requests to K. Schrör at the above address     

Inhibitory effects of mesoionic 3-aryl substituted oxatriazole-5-imine derivatives on vascular smooth muscle cell mitogenesis and proliferation in vitro

1. The effects of oxatriazole-type (GEA 3162 and GEA 5624) nitric oxide (NO) donors on mitogenesis and proliferation were studied in vascular smooth muscle cell (VSMC) culture. The effects of the GEA-compounds were compared with well-known NO-donors 3-morpholinosydnonimine (SIN-1) and S-nitroso-N-acetylpenicillamine (SNAP).
2. All NO-donors released NO and increased the production of cyclic GMP concentration-dependently. The production of cyclic GMP was inhibited by the guanylate cyclase inhibitor, ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one).
3. The NO-donors inhibited basal and serum-induced DNA synthesis concentration-dependently. The GEA-compounds were needed in concentrations 10 times lower than SIN-1 and SNAP. GEA 3162, SIN-1 and SNAP were also able to inhibit serum-induced cell proliferation. GEA 5624 was ineffective. The antimitogenic effect of NO-donors was not reduced by inhibiting the guanylate cyclase.
4. These results suggest that NO inhibits serum-induced DNA synthesis and proliferation of VSMC by a cyclic GMP-independent mechanism. The oxatriazole-type NO-donor GEA 3162 was found to be a more potent inhibitor of mitogenesis and cell proliferation than SIN-1 and SNAP.

     

Characterization of the transport of uracil across Caco-2 and LLC-PK1 cell monolayers

Purpose. The purpose of this study was to characterize the transport of uracil, a pyrimidine nucleobase, in Caco-2 and LLC-PK₁ cells. Methods. Caco-2 and LLC-PK₁ cells were grown to confluency on a permeable polycarbonate membrane insert to permit transport and uptake experiments after the loading of uracil on either the apical or basolateral side. Results. The vectorial transport of uracil in both directions was saturable with comparable K_m and V_max in Caco-2 cell monolayers, probably because of a Na⁺-independent transport system located on the basolateral membrane. In LLC-PK₁ cell monolayers, two distinct transport systems, namely a Na⁺-dependent and a Na⁺-independent, were functional in the apical to basolateral (A-B) transport of uracil. The first system was saturable with a K_m value of 3.67 ± 0.40 M, a V_max of 11.31 ± 0.91 pmol/cm²/min, and a Na⁺:uracil coupling stoichiometry of 1.28 ± 0.20. The second system was Na⁺ independent and satuable with a low affinity (K_m, 50.37 ± 9.61 M) and V_max (16.01 ± 4.48 pmol/cm²/min). The two transport systems appeared to be located on the apical membrane. Conclusion. The mechanism of uracil transport differs depending on cell lines; a Na⁺-independent system on the basolateral membrane in Caco-2 cells and both Na⁺-dependent and Na⁺-independent systems on the apical membrane in LLC-PK₁ cells seem to be responsible for the difference.     

The effects of the nitric oxide donors molsidomine and SIN-1 on human polymorphonuclear leucocyte functionin vitro andex vivo

Summary The nitrovasodilator and nitric oxide donor molsidomine and its metabolite SIN-I dilate vascular smooth muscle and inhibit platelet activation by increasing intracellular concentrations of cyclic GMP We have therefore studied the effects of molsidomine and SIN-I on isolated human polymorphonuclear leucocytes (PMN)in vitro andex vivo. In vitro molsidomine dose-dependently reduced-glucuronidase release and the generation of superoxide anions from non-activated and from FMLP- or PAF-stimulated human PMNs. SIN-1 was equally effective in reducing (-glucuronidase release and totally inhibited oxygen radical generation at a concentration of 580 mol · l^–1.In a double-blind, placebo-controlled, randomized trial we also studied-glucuronidase release and the generation of superoxide anions from isolated PMNs. Blood was drawn from 12 healthy volunteers before and 3 h after oral molsidomine (16 mg) or placebo. There was no statistically significant difference in-glucuronidase release and superoxide anion formation when the PMNs were isolated before or after molsidomine or placebo. This was the case for non-activated, as well as FMLP- or PAF-stimulated PMNs.Thus, the nitric oxide donors molsidomine and its metabolite SIN-I caused a dose-dependent inhibition of PMN functionsin vitro, but no significant inhibition when the PMNs were isolated after oral molsidomine.     

Cross-tolerance to L-arginine-dependent guanylate cyclase activators in nitrate-tolerant LLC-PK1 kidney epithelial cells

Recently, it was shown that in LLC-PK1 kidney epithelial cells hormones such as vasopressin or oxytocin increase cyclic GMP in a receptor-mediated and L-arginine-dependent manner. In the present study, the possible existence of cross-tolerance to vasopressin and oxytocin was investigated in nitrate-tolerant LLC-PK1 cells. Pretreatment with 1 mM glyceryl trinitrate for 3 h decreased cyclic GMP stimulation by 1 microM vasopressin and 1 microM oxytocin by 49% and 54%, respectively. Under the same conditions, cyclic GMP stimulation at 1 microM sodium nitroprusside was diminished by 56% whereas the cyclic GMP response to 100 microM glyceryl trinitrate was virtually abolished. Our results demonstrate that a substantial degree of cross-tolerance to L-arginine-dependent guanylate cyclase activators occurs in nitrate-pretreated nonvascular cells which may be due to glyceryl trinitrate-induced desensitization of soluble guanylate cyclase.     

Effect of 3-morpholinosydnonimine (SIN-1) and NG-nitro-l-arginine (NNA) on isolated perfused anaphylactic guinea-pig hearts

Summary The modulating effects of exogenous and endogenous nitric oxide (NO) on the cardiac anaphylactic reaction and eicosanoid release were investigated in isolated perfused sensitized guinea-pig hearts using 3-morpholinosydnonimine (SIN-1), the active metabolite of molsidomine, as NO-donor and N^G-nitro-l-arginine (NNA) as an inhibitor of NO biosynthesis. Infusion of SIN-1 (final concentrations in the perfusates 0.3 or 1.0 mmol/l) elevated coronary flow under basal conditions as well as during cardiac anaphylaxis, while NNA (0.1 mmol/l) decreased basal coronary flow and aggravated the anaphylactic coronary constriction. Both drugs did not modify the characteristic biphasic profile of the coronary constriction after antigen challenge with an initial more severe phase followed by a less pronounced long-lasting flow reduction. Neither SIN-1 nor NNA affected spontaneous heart rate. However, while NNA tended to prolong the duration of antigen-induced arrhythmias, SIN-1 (1 mmol/l) had an inhibitory effect. This protection might be related to the increased coronary flow in the presence of SIN-1. SIN-1 inhibited anaphylactic release of cysteinyl-leukotrienes (LT) and 6-keto-prostaglandin (PG) F₁, but did not influence thromboxane (TX) B₂ release. On the other hand, NNA (0.1 mmol/l) inhibited anaphylactic release of TXB₂, but had only marginal effects on the release of cysteinyl-LT and 6-keto-PGF₁. The results suggest that exogenous and endogenous NO functionally antagonize the effects of vasoconstrictor mediators released after antigen challenge. Additional effects of high concentrations of SIN-1 and NNA on antigen-induced eicosanoid release could modulate the vascular actions of these drugs during cardiac anaphylaxis. Send offprint requests to K. I. Thelen at the above address     

Total prevention of the development of in vitro tolerance to organic nitrates. Experiments with antioxidants.

The nearly total inhibition of development of pharmacological tolerance to an organic nitrate is reported here for the first time. The development of in vitro tolerance in the rabbit aorta to isosorbide-5-mononitrate (CAS 87-33-2) was potently inhibited by five structurally unrelated antioxidants--diaminodurol, ascorbic acid, potassium sulphite, pyrogallol and quercetin. Diaminodurol, ascorbic acid and potassium sulphite decreased, but quercetin increased, the spasmolytic activity of isosorbide-5-mononitrate. Diaminodurol, potassium sulphite, quercetin and ascorbic acid potently inhibited the spasmolytic activity of nitric oxide (NO). Quercetin also inhibited the development of in vitro tolerance to glyceryl trinitrate. It is suggested that tolerance to organic nitrates is the result of biochemical damage caused by a reactive intermediate such as NO. To test this possibility directly the effect of pretreatment with NO on the spasmolytic activity of glyceryl trinitrate (CAS 55-63-0) was examined. This pretreatment produced a small but significant tolerance to glyceryl trinitrate and to SIN-1 (3-morpholinosydnone imine), which also acts through guanylate cyclase. There was no effect on the activity of the unrelated vasodilators nitrendipine and theophylline. It is concluded that the reaction between NO and soluble quanylate cyclase is a real but minor cause of tolerance to organic nitrates. Other possible mechanisms of tolerance development are discussed.     

Cyclic GMP mediates SIN-1-induced inhibition of human polymorphonuclear leukocytes.

Different nitrovasodilators were used to assess the role of cyclic GMP in the regulation of polymorphonuclear leukocyte (PMN) function. Molsidomine and its metabolites, 3-morpholinosydnonimine (SIN-1) and N-nitroso-N-morpholinoaminoacetonitrile (SIN-1A) at 0.01-1 mM, inhibited lysosomal enzyme release from PMN stimulated by 30 nM formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP). At 1 mM, molsidomine, SIN-1 and SIN-1A decreased beta-glucuronidase release by 19, 37 and 46% of the control, respectively. Glyceryl trinitrate (GTN) and sodium nitroprusside (SNP) showed no effect on beta-glucuronidase release from PMN. At 1 mM, SIN-1A, SIN-1 and SNP in the presence of 0.5 mM isobutylmethylxanthine (IBMX) stimulated cyclic GMP 21-, 9- and 14-fold, respectively, demonstrating a relation between cyclic GMP stimulation and neutrophil inhibition by the molsidomine metabolites. GTN and unmetabolized molsidomine were without effect on cyclic GMP levels. The hypothesis of an inhibitory effect of cyclic GMP on neutrophil function was further supported by the attenuation of SIN-1-induced inhibition of enzyme release by methylene blue (10 microM), an inhibitor of soluble guanylate cyclase. Moreover, 8-bromo cyclic GMP and dibutyryl cyclic GMP, 1 mM, decreased beta-glucuronidase release from FMLP-stimulated PMN by 12 and 44% of the control, respectively. These data demonstrate that cyclic GMP is an inhibitory second messenger in human PMN and suggest that this action of SIN-1 may be of considerable interest under conditions of platelet/PMN activation, e.g. during myocardial ischemia.     

L-cysteine augments the vasorelaxation induced by sodium nitrite and SIN-1 but not that due to acetylcholine.

The effects of 1 mM L-cysteine on sodium nitrite-, 3-morpholinosydnonimine (SIN-1)- and acetylcholine-induced relaxation and cyclic GMP accumulation were studied in isolated noradrenaline-precontracted rat mesenteric arterial rings. L-Cysteine augmented the relaxation and cyclic GMP increase induced by sodium nitrate and SIN-1 but not those induced by acetylcholine. The effects of L-cysteine on relaxation were independent of the presence of intact endothelium. The results suggest that L-cysteine protects exogenously released nitric oxide.     

Nitric oxide regulation of monkey myometrial contractility

1. We evaluated the effect of the nitric oxide (NO) donor CysNO (S-nitroso-L-cysteine) and endogenous NO upon spontaneous contractility in non-pregnant cynomolgus monkeys. We also assessed the role of intracellular guanosine 3′,5′-cyclic monophosphate ([cyclic GMP]_i) as a second messenger for NO in monkey uterine smooth muscle.
2. CysNO reduced spontaneous contractility by 84% (P<0.05) at maximal concentrations, and significantly elevated [cyclic GMP]_i (P<0.05). However, increases in [cyclic GMP]_i were not required for CysNO-induced relaxations; CysNO inhibited contractile activity despite the complete inhibition of guanylyl cyclase by methylene blue or LY83,583.
3. Analogues of cyclic GMP had no significant effect upon spontaneous contractile activity. L-arginine produced a 62% reduction in spontaneous activity (P<0.05) while D-arginine had no effect. The competitive nitric oxide synthase (NOS) inhibitor N^ω-nitro-L-arginine (L-NOARG) not only blocked L-arginine-induced relaxations, but also significantly increased spontaneous contractile activity when added alone (P<0.05); the inactive D-enantiomer of NOARG had no such effect.
4. While both endogenous NO and the NO donor CysNO relax monkey myometrium, this effect is not causally related to CysNO-induced elevations in [cyclic GMP]_i. The failure of cyclic GMP analogues to alter monkey uterine smooth muscle tension also argues against a role for [cyclic GMP]_i in the regulation of uterine contractility. Not only do these findings argue for the existence of a functionally-relevant NOS in the monkey uterus, but increases in contractile activity seen in the presence of NOS inhibitors suggest a role for NO in the moment-to-moment regulation of contractile activity in this organ.

     

Characterization of LLC-PK1 Kidney Epithelial Cells as an in Vitro Model for Studying Renal Tubular Reabsorption of Protein Drugs

Purpose. The purpose of this study was to assess whether LLC-PK₁ renal epithelial cells could serve as an in vitro model for studying the renal tubular reabsorption of protein drugs. Methods. The association of ¹¹¹In-labeled model protein drugs, bovine serum albumin (BSA), superoxide dismutase (SOD), soybean trypsin inhibitor (STI), and [Asu^1,7]-eel calcitonin (Asu-ECT), with the monolayers of LLC-PK₁ renal epithelial cells was characterized under various conditions. Results. The cellular association of these proteins was temperature-dependent and varied according to the protein. Saturation kinetics were observed for STI association, with the apparent Km and Vmax values determined to be 66.3 µg/ml and 250 ng/mg protein/min, respectively. The association of STI decreased with increases in medium pH from 5.4 to 8.4 and was inhibited significantly by 2,4-dinitrophenol, sodium azide, cytochalasin B, and colchicine, suggesting that the cellular association involved endocytosis. Mutual inhibition was observed in competitive binding experiments with the four protein drugs, suggesting that they shared a common binding site on the luminal membrane of LLC-PK₁ cells. Taken together, these findings show that a variety of protein drugs bind to LLC-PK₁ cells in a non-specific manner and possibly undergo endocytosis, a phenomenon that is similar to in vivo proximal tubular reabsorption. Conclusions. LLC-PK₁ renal epithelial cells would be a suitable model system for the study of the renal proximal tubular reabsorption of protein drugs.     

Venous plasma levels of endothelin-1 are not altered immediately after nitroglycerin infusion in healthy subjects

Endothelin-1 and nitric oxide play an important regulatory role in the control of vascular smooth muscle tone. Nitroglycerin (NTG), a nitric oxide donating drug, may inhibit endothelin production. In this double-blind placebo-controlled crossover study, plasma levels of endothelin-1 were measured before and immediately (5–30 s) after 80 min infusion of NTG (glyceryl trinitrate) or saline in 12 healthy subjects. On two different days separated by at least 1 week, NTG in four different doses, 0.015, 0.25, 1.0, and 2.0 g·kg^–1·min^–1, or placebo (isotonic saline) was infused successively for 20 min each dose. During the infusion blood pressure and heart rate were measured. NTG infusion significantly decreased systolic blood pressure from 112.4 to 103.4 mmHg and pulse pressure from 39.3 to 29.5 mmHg. Heart rate increased from 62.7 to 73.1 beats·min^–1. No changes in endothelin-1 plasma levels were induced by NTG infusion (2.4 pg·ml^–1 before NTG vs. 2.7 pg·ml^–1 after NTG) and placebo infusion also did not affect plasma endothelin-1. It is concluded that venous plasma levels of endothelin-1 are not altered immediately after NTG infusion.     

Comparative effects of activation of soluble and particulate guanylyl cyclase on cyclic GMP elevation and relaxation of bovine tracheal smooth muscle.

1. The effects of nitric oxide-donating compounds and atrial natriuretic peptide on cyclic GMP accumulation and mechanical tone were compared with the effects of isoprenaline in bovine tracheal smooth muscle. 2. Sodium nitroprusside, glyceryl trinitrate, S-nitroso-N-acetylpenicillamine (SNAP), atrial natriuretic peptide and isoprenaline each caused concentration-dependent inhibitions of histamine-maintained tone (EC50 values 320 +/- 80, 150 +/- 45, 14,000 +/- 4,000, 2.8 +/- 0.8 and 6.6 +/- 4.3 nM respectively). 3. When compared with their effects on histamine-induced tone, sodium nitroprusside was equally potent and effective in causing relaxation of methacholine-supported tone (EC50 290 +/- 90 nM) while isoprenaline was as effective, but less potent (EC50 30 +/- 7 nM). SNAP was more potent and equi-effective as a relaxant of methacholine-supported tone (EC50 340 +/- 140 nM). At the maximum concentrations of glyceryl trinitrate and atrial natriuretic peptide tested against methacholine-supported tone, relaxations of 52% and 14% of the isoprenaline maximum were seen. 4. Sodium nitroprusside, glyceryl trinitrate and atrial natriuretic peptide each induced concentration-dependent increases in cyclic GMP accumulation. The time-courses of accumulation correlated closely with the relaxant actions of these compounds. 5. Pretreatment of tracheal smooth muscle with sodium nitroprusside or SNAP caused a rightward shift of the concentration-effect curve for histamine while reducing the maximum response. 6. LY 83583, a putative guanylyl cyclase inhibitor, caused a concentration-dependent reduction in basal cyclic GMP accumulation in tracheal smooth muscle and inhibited the effects of sodium nitroprusside on cyclic GMP accumulation. 7. LY 83583 also inhibited the relaxation of histamine-supported tone by glyceryl trinitrate, sodium nitroprusside, SNAP and atrial natriuretic peptide, and also sodium nitroprusside- and SNAP-induced relaxation of methacholine-supported tone. However, it had no significant effect on glyceryl trinitrate-induced relaxation of methacholine-supported tone. 8. It is concluded that the relaxant actions of sodium nitroprusside, glyceryl trinitrate, SNAP and atrial natriuretic peptide follow as a result of their ability to activate either soluble or particulate guanylyl cyclase leading to cyclic GMP accumulation. Although there does not seem to be any functional difference in the relaxant response to cyclic GMP generated by the particulate as opposed to soluble form(s) of guanylyl cyclase, atrial natriuretic peptide receptor/guanylyl cyclase activation was much less effective in causing relaxation of methacholine-supported tone when compared to activators of soluble guanylyl cyclase.