Krebs-Henseleit Saline Research Articles

Vasorelaxation of goat mesenteric artery is mediated by endothelial Na(+)-K(+)-ATPase.

Objective:To examine the role of Na+-K+-ATPase and K+ channels in mediating vasorelaxation in the superior mesenteric artery of Capra hircus.Materials and Methods:Goat superior mesenteric artery (GSMA) was cut into 1.5–2 mm circular rings and mounted in a thermostatically controlled (37°C ± 0.5°C) organ bath containing 20 ml of modified Krebs-Henseleit saline (MKHS) (pH 7.4), with continuous aeration under 1.5 g tension for 90 min. Endothelium-intact (ED+) or endothelium-denuded (ED−) GSMA ring was contracted with phenylephrine (PE) or 5-hydroxytryptamine (5-HT) (1 μM–0.1 mM) in the absence or presence of ouabain (0.1 μM). KCl (1 μM–10 mM) was added cumulatively to K+-free MKHS-pre-contracted (ED+/−) rings in the absence or presence of ouabain (0.1 μM) or barium (1 μM) or 4-aminopyridine (1 μM).Results:Ouabain did not alter the basal tone of the arterial ring. The contractile response induced by PE (Emax: 50.46 ± 2.68, pD2: 5.53 ± 0.04) and 5-HT (Emax: 30.86 ± 1.33, pD2: 6.17 ± 0.03) in ED+ ring was significantly (P < 0.001) augmented in ED− rings (PE: Emax: 93.30 ± 2.11, pD2: 6.41 ± 0.04; 5-HT: Emax: 95.07 ± 0.99, pD2: 6.27 ± 0.03). The contractile response induced by PE and 5-HT in ED+ or ED− rings in the presence of ouabain was almost identical with that of ED− rings. Vasorelaxation of KCl (Emax: 2.90 ± 1.14, pD2: 3.9 ± 0.03) was significantly attenuated in the presence of ouabain (Emax: 73.8 ± 5.16, pD2: 4.3 ± 0.04), Ba2+ (Emax: 16.34 ± 4.7, pD2: 3.22 ± 0.02), 4-AP (Emax: 18.16 ± 2.4, pD2: 3.68 ± 0.03), ouabain and Ba2+ (Emax: 70.09 ± 3.66, pD2: 4.41 ± 0.04), and ouabain and 4-AP (Emax: 66.98 ± 4.61, pD2: 4.13 ± 0.06).Conclusion:The vasorelaxation in GSMA is mediated by the endothelium-derived hyperpolarizing factor (EDHFs) such as ouabain-sensitive Na+-K+-ATPase, KIR and Kv channels.

Biphasic effects of angiotensin (1-7) and its interactions with angiotensin II in rat aorta.

Using isolated rat aortic rings perfused with Krebs-Henseleit saline, the vascular effects of angiotensin (1-7) (Ang [1-7]) and its interactions with angiotensin II (Ang II) were investigated. Ang (1-7) induced endothelium-dependent relaxation and vasodilating effects in preparations precontracted with phenylephrine. Without preconstriction, Ang (1-7) at high doses (10(-6) 10(-5) M) produced either a significant inhibition of Ang II-induced vasoconstriction or a non-tachyphylactic vasopressor response. While losartan inhibited the vasoconstriction induced by Ang (1-7), A779 blocked only its relaxation. Unlike losartan, blockade of AT(2)-receptors with PD 123319 had no effect. Taking into account the biphasic effects of angiotensin (1-7), we propose that it is one of the active components of the renin-angiotensin system, which is involved as a modulator both in the counter-regulatory actions of Ang II and in the self-regulation of its own vasodilating effects.

The association between meconium and the production and reabsorption of lung liquid and lactate loss by in vitro lungs from fetal guinea pigs

Objective: We sought to use an in vitro approach to determine relationships between meconium and fetal lung liquid production and to relate meconium to possible problems in lung liquid removal at birth. Study Design: Near-term fetal guinea pigs were divided according to the level of spontaneously occurring meconium (no meconium, light meconium, or heavy meconium). Their lungs were maintained in vitro in Krebs-Henseleit saline solution for 3 hours. Lung liquid production or reabsorption was measured by a dye-dilution method, and loss of lactate to the lung liquid and outer Krebs-Henseleit solution was monitored. Reabsorptions were investigated by activating powerful responses with dinitrophenol during the middle hour. Results: During the first hour, lungs from meconium-free fetuses produced liquid at 1.25 ± 0.12 mL/kg–1 body weight · h–1; rates from light or heavy meconium groups were not significantly different (n = 18). Similarly, total lactate loss was not significantly different between the meconium-free and light-meconium groups but was twice as high in heavy-meconium preparations (73.68 ± 10.60 μmol/L · g–1 dry lung tissue · h–1; P <.025, analysis of variance). The meconium-free and heavy-meconium groups continued to produce fluid, with no significant change throughout the 3 hours of incubation; lactate losses fell slightly. Therefore there were no problems with fluid production with meconium present, but the high-lactate losses with heavy meconium suggested long-term intrauterine hypoxia. Dinitrophenol produced powerful reabsorptions in lungs from meconium-free fetuses (–0.85 ± 0.35 mL · kg–1 body weight · h–1; P <.005, analysis of variance; P <.0005, regression analysis) but failed to do so in heavy-meconium fetuses (n = 36). Lactate losses rose 2-fold in both groups (P <.005 to P < 0.0005, analysis of variance and regression analysis), despite already elevated losses with heavy meconium (n = 12). Therefore, in heavy-meconium fetuses, dinitrophenol affected metabolic pathways but did not activate fluid reabsorption, suggesting damage to reabsorptive mechanisms. Conclusion: Unless major airways are blocked, meconium is not associated with reduced fetal lung liquid production, which can cause poor lung development, but there may well be poor fluid removal after birth because of compromised reabsorptive mechanisms, which are unlikely to be helped by possible hormonal intervention. (Am J Obstet Gynecol 2000;183:235-44.)

The association between meconium and the production and reabsorption of lung liquid and lactate loss by in vitro lungs from fetal guinea pigs*1

Objective: We sought to use an in vitro approach to determine relationships between meconium and fetal lung liquid production and to relate meconium to possible problems in lung liquid removal at birth. Study Design: Near-term fetal guinea pigs were divided according to the level of spontaneously occurring meconium (no meconium, light meconium, or heavy meconium). Their lungs were maintained in vitro in Krebs-Henseleit saline solution for 3 hours. Lung liquid production or reabsorption was measured by a dye-dilution method, and loss of lactate to the lung liquid and outer Krebs-Henseleit solution was monitored. Reabsorptions were investigated by activating powerful responses with dinitrophenol during the middle hour. Results: During the first hour, lungs from meconium-free fetuses produced liquid at 1.25 ± 0.12 mL/kg–1 body weight · h–1; rates from light or heavy meconium groups were not significantly different (n = 18). Similarly, total lactate loss was not significantly different between the meconium-free and light-meconium groups but was twice as high in heavy-meconium preparations (73.68 ± 10.60 μmol/L · g–1 dry lung tissue · h–1; P <.025, analysis of variance). The meconium-free and heavy-meconium groups continued to produce fluid, with no significant change throughout the 3 hours of incubation; lactate losses fell slightly. Therefore there were no problems with fluid production with meconium present, but the high-lactate losses with heavy meconium suggested long-term intrauterine hypoxia. Dinitrophenol produced powerful reabsorptions in lungs from meconium-free fetuses (–0.85 ± 0.35 mL · kg–1 body weight · h–1; P <.005, analysis of variance; P <.0005, regression analysis) but failed to do so in heavy-meconium fetuses (n = 36). Lactate losses rose 2-fold in both groups (P <.005 to P < 0.0005, analysis of variance and regression analysis), despite already elevated losses with heavy meconium (n = 12). Therefore, in heavy-meconium fetuses, dinitrophenol affected metabolic pathways but did not activate fluid reabsorption, suggesting damage to reabsorptive mechanisms. Conclusion: Unless major airways are blocked, meconium is not associated with reduced fetal lung liquid production, which can cause poor lung development, but there may well be poor fluid removal after birth because of compromised reabsorptive mechanisms, which are unlikely to be helped by possible hormonal intervention. (Am J Obstet Gynecol 2000;183:235-44.)

Effects of lung expansion on lung liquid production in vitro by lungs from fetal guinea-pigs. II. Evidence for generation of an inhibitory factor.

Lungs from near-term fetal guinea-pigs were supported in vitro for 3 h; lung liquid production was measured by a dye-dilution method using Blue Dextran 2000 [fetuses 63 +/- 2 days of gestation, 97.6 +/- 19.8 (SD) g body weight]. Preparations were incubated in pairs taken from the same mother. Twenty lungs incubated in pairs without treatment (controls) showed no significant changes in fluid production throughout incubation (analysis of variance; regression analysis); rates in successive hours were: first lung, 1.36 +/- 0.39, 1.09 +/- 0.34 and 1.27 +/- 0.42 ml/kg body weight per h; second lung, 1.46 +/- 0.52, 1.09 +/- 0.41 and 1.18 +/- 0.43 ml/kg body weight per h. Twenty lungs were incubated similarly in pairs, but after one hour one lung from each pair was expanded with Krebs-Henseleit saline in volumes approximating those of the first breath (68 +/- 10% of lung volume). The expanded lungs began to reabsorb fluid immediately after expansion; the untreated lungs also stopped production or reached reabsorption by the final hour. Rates in successive hours were: expanded lungs; before expansion, 1.00 +/- 0.21, after expansion, -0.23 +/- 0.17 and 0.14 +/- 0.09 ml/kg body weight per h; unexpanded lungs, 1.27 +/- 0.49, 0.02 +/- 0.01 and -0.01 +/- 0.004 ml/kg body weight per h. The decrease in production was significant for each type of lung. The effects persisted in both expanded and unexpanded lungs in the presence of 1.78 x 10(-5) M phentolamine (n = 12; 70 +/- 2% expansion). The results suggest that expansion of the lungs at birth may release an unknown inhibitory factor, provisionally termed Expansion Factor (EF), within the lungs; this agent, probably not a catecholamine, can change lung fluid production into reabsorption and may partly account for the failure of beta-antagonists to prevent fluid reabsorption at delivery.

Overnight storage of the porcine isolated splenic artery enhances endothelium-dependent contractions to NG-nitro-L-arginine methyl ester without impairing endothelium-dependent dilator function.

This study examined the influence of overnight storage on endothelium-independent contractions to 5-hydroxytryptamine (5-HT), endothelium-dependent contractions to NG-nitro-L-arginine methyl ester (L-NAME), and endothelium-dependent relaxations to substance P (SP) and L-arginine, using the porcine isolated splenic artery. In endothelium-intact (E+) segments from fresh porcine isolated splenic arteries or segments from the same vessels stored overnight at 4 degrees C, either in Krebs-Henseleit saline or in Krebs-Henseleit saline containing 1 mM L-arginine, 5-HT caused concentration-related contractions that were similar under all three conditions. Overnight storage enhanced contractions of the splenic artery to L-NAME, an effect not observed if the vessels were co-stored with 1 mM L-arginine. L-NAME failed to contract endothelium-denuded (E-) segments from fresh tissues or tissues stored overnight, indicating that its constrictor effects were endothelium-dependent. SP caused concentration-related, endothelium-dependent relaxations of the splenic artery that were inhibited by 100 microM L-NAME, indicating that the relaxations could be attributed to the stimulated release of NO from endothelial cells. Established contractions to 100 microM L-NAME in E+ segments from fresh tissues, or segments from the same tissues stored overnight at 4 degrees C, either in Krebs-Henseleit saline or in Krebs-Henseleit saline containing 1 mM L-arginine, were all reversed by 1 mM L-arginine to similar extents, indicating that overnight storage did not affect endothelium-dependent dilator responses to L-arginine.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for functional dissociation of dependence and tolerance in guinea‐pig isolated ileal segments following 20 hour exposure to morphine in vitro

1. In the present study we have examined the relationship between tolerance and dependence in isolated ileal segments from the guinea-pig under three different conditions: fresh preparations not previously exposed to morphine (fresh/morphine naive); preparations stored overnight at 4 degrees C in modified Krebs-Henseleit saline (overnight-stored/morphine-naive); preparations stored overnight at 4 degrees C in Krebs-Henseleit saline containing 10 microM morphine and extensively washed with modified Krebs-Henseleit saline to remove residual morphine (overnight-stored/morphine-exposed). 2. Morphine produced a concentration-dependent inhibition of the response of ileal segment to 0.1 Hz, 1 ms and 10 V transmural field stimulation in fresh/morphine-naive, overnight-stored/morphine-naive and overnight-stored/morphine-exposed preparations. The maximum effect observed was similar in all three preparations-approximately 80% inhibition. Although, morphine was significantly more potent in the fresh/morphine-naive preparations (pD2 6.72 +/- 0.05, n = 8) than either the overnight-stored/morphine-native (pD2 6.42 +/- 0.11, n = 8) or the overnight-stored/morphine-exposed (pD2 6.44 +/- 0.14, n = 8), there was no significant difference between the overnight exposure to ileal segments to 10 microM morphine at 4 degrees C failed to induce tolerance to morphine. 3. The mu opiate receptor antagonist, naloxone (10 microM), produced contractions in both fresh/morphine-naive and overnight-stored/morphine-naive ileal segments following acute exposure to 10 microM morphine. Naloxone (10 microM) also produced contractions in 2/9 fresh/morphine-naive, 1/9 overnight-stored/morphine-naive and 7/9 overnight-stored/morphine-exposed preparations in the absence of morphine. The greater incidence of naloxone-induced contractions in overnight-stored/morphine-exposed preparations,suggests that dependence in this model is the product of adaptive changes that outlive the presence of morphine.4. The selective alpha2-adrenoceptor agonists, clonidine (0.3 microM) and 5-bromo-6-[2-imidazolin-2-ylamino]-quinoxaline bitartrate (UK-14304, 1 microM), inhibited naloxone-induced contractions in overnight-stored/morphine-exposed preparations of ileal segments (n = 4 preparations for each agonist), suggesting that the response is due to transmitter release from the myenteric plexus.5. The findings in the present study indicate that tolerance and dependence to morphine in ileal segments of the guinea-pig can be functionally dissociated by overnight exposure to morphine at 4 degrees C.The development of tolerance to morphine, unlike dependence, appears to be a temperature-dependent process. This also raises the possibility that naloxone possesses intrinsic negative agonism at morphine sensitive receptors, which is manifested as a functional response only after adaptive changes in the myenteric plexus following exposure to morphine.

Mitogen-activated protein (MAP) kinase stimulation by phorbol esters and external load in the isolated perfused heart.

Mitogen-activated protein kinases are a family of serine/ threonine kinases that have been implicated in a wide variety of cellular processes [for review see 1,2]. These kinases are thought to participate in a protein kinase cascade leading to cell growth as they have been shown to phosphorylate ribosomal protein S6 and various transcription factors. In response to most growthpromoting and mitogenic factors tested, as well as to many other signals such as phorbol esters, MAP kinase phosphorylation is increased on both threonine and tyrosine residues, leading to stimulation of its kinase activity [3-51. Phosphorylation of MAP kinase involves an activator which is itself a kinase, with dual specificity for both tyrosine and serindthreonine [6]. Recent reports suggest that MAP kinase is involved in the signalling events leading to heart hypertrophy [7,8]. To evaluate this hypothesis further, we have investigated the effects of hypertrophic agents on the activity of MAP kinase in isolated perfused heart preparations. As hypertrophic agents, TPA and high load were used as they are both known to induce responses characteristic of hypertrophy in rat heart preparations. Hearts were perfused retrogradely with Krebs-Henseleit saline (1 19 mM NaCI, 25 mM NaHCO,, 4.7 mM KCl, 2.5 mM CaCI,, 1.2 mM MgSO,, 1.2 mM KH,PO, and 10 mM glucose, equilibrated with 95% 45% C02, pH 7.6 at 37°C). For control experiments, perfusions were carried out at for 15 min at 5.5 kPa. After that, hearts were either exposed to TPA or high aortic pressure for a further 5 min period. When present, TPA was added in the perfusion medium at a final concentration of 1pM. For the experiments at high aortic pressure, the perfusion pressure was increased to 16.3 kPa (120 mm Hg). Hearts were extracted with 20mM j3-glycerophosphate pH 7.5, containing 20mM NaF, 2mM EDTA, 0.2mM Na,VO,, lOmM benzamidine, 25 pg/ml leupeptin, 50 pglml PMSF and 0.3% (v/v)J?-mercaptoethanol and centrifuged for lOmin at 1oooOxg. The supernatant was applied to a Mono Q FPLC column equilibrated with 50 mM Tris-HC1 pH 7.5, containing 2 mM EDTA, 2 mM EGTA, 0.1% Jmercaptoethanol, 5% glycerol, 0.03% Brij 35, 0.3 mM Na,VO,, ImM benzamidine and 4 pg/ml leupeptin. Myelin basic protein (MBP) kinase activity was eluted with linear gradient of NaCl (00.3 M) and measured as described previously [7]. FPLC of the heart extracts revealed two peaks of MBP kinase activity that were stimulated with exposure to TPA. The first peak eluted at 0.16 M NaCl and the activity was stimulated %fold compared with control. The second peak eluted at 0.22 M NaCl and the activity was stimulated 2.3-fold (Fig. 1). Extracts of hearts exposed to high aortic pressures showed an identical elution profile but stimulation was lower (2-fold for the first peak and 1.6-fold for the second peak) (Fig. 1). Using a kinase renaturation assay method [9], we examined the peak fractions from FPLC of TPA-treated hearts for their ability to phosphorylate MBP in gels in siru. We found that the molecular masses were 42kDa and 44kDa for the first and second peak respectively (Fig. 2a). The same method was used to examine the crude extracts of control and TPA-treated hearts. MBP kinase

Some effects of different extracellular proteins on oxygen consumption and heat production in isolated rat hepatocytes

When hepatocytes prepared from 24-h-fasted rats were washed, suspended and incubated in Krebs-Henseleit bicarbonate-buffered saline, the endogenous rates of O2 consumption and heat production were 2.13 +/- 0.13 mumol/min per g wet wt. and 1.00 +/- 0.05 J/min per g wet wt. respectively. The inclusion of 2.5% (w/v) defatted and dialysed bovine serum albumin in either the cell suspension (washing) buffer or the cell incubation buffer produced a 20-25% increase in O2 consumption and heat production: these rates were increased by an additional 20-25% when the albumin (2.5%) was present in both the cell suspension and the cell incubation buffers. There was an inverse relationship between the increases in O2 consumption and heat production and the leakage of lactate dehydrogenase from the isolated hepatocytes: the inclusion of purified bovine serum albumin decreased lactate dehydrogenase leakage from 40% to 15% of total enzyme content. The calorimetric-respirometric ratios for hepatocytes incubated both in the absence (-461 +/- 19 kJ/mol O2) and presence (-477 +/- 8 kJ/mol O2) of the purified protein are very similar to the theoretical, thermochemically derived oxycaloric equivalents.

An examination of the sources of calcium for contractions mediated by postjunctional α1‐ and α2‐adrenoceptors in several blood vessels isolated from the rabbit

1. The roles of intracellular and extracellular-derived Ca2+ in alpha-adrenoceptor-mediated contractions to noradrenaline (NA) have been investigated in several isolated blood vessels from the rabbit by examining responses in the presence of a modified Krebs-Henseleit saline with 2.5 mM Ca2+ and a Ca2(+)-buffered saline with 0.1 microM free Ca2+. 2. NA was tested in preparations of the abdominal aorta, distal saphenous artery, renal vein, lateral saphenous vein, plantaris vein and ear vein exposed to a Ca2(+)-buffered saline with 0.1 microM [Ca2+]. A concentration of NA which was maximally effective in modified Krebs-Henseleit saline, produced an initial transient contraction (ITC) followed by a relaxation towards baseline. This is evidence that alpha-adrenoceptor-mediated responses in all these blood vessels depend upon calcium from both sources. 3. The ITC was particularly pronounced in the arteries and was associated more closely with the alpha 1-receptor subtype. In the abdominal aorta, distal saphenous artery and renal vein the ITC can almost exclusively be attributed to an alpha 1-adrenoceptor (prazosin-sensitive, rauwolscine-resistant). In the ear vein, and to a lesser extent the plantaris vein, the ITC was mediated in part by an alpha 2-adrenoceptor (prazosin-resistant, rauwolscine-sensitive). 4. alpha 2-Adrenoceptors in the lateral saphenous vein largely account for the response to NA in modified Krebs-Henseleit saline, but alpha 1-adrenoceptors mediate the ITC in Ca2(+)-buffered saline. After selective inactivation of alpha 1-adrenoceptors with a combination of phenoxybenzamine and rauwolscine, responses to NA in modified Krebs-Henseleit saline are slow in onset and there is no ITC in Ca2(+)-buffered saline. 5. The possible significance of the coupling of postjunctional alpha 2-adrenoceptors to dual sources of Ca2 + is discussed in relation to the interaction between alpha-adrenoceptor subtypes and the ease of demonstrating functional alpha 2-adrenoceptors in isolated blood vessels.

Relationship between intracellular ATP and the sodium pump activity in dog renal tubules

To examine the potential effect of the cellular ATP concentration and of the phosphate potential on the function of the sodium pump in intact renal cells, the ATP content of dog cortical tubules was first modified by a 30-min preincubation with one of the following effectors: 5 or 10 mM fructose, 2.5 mM adenosine 5'-monophosphate (AMP), or 2.5 mM adenosine in the presence of substrates (10 mM glutamine + 1 mM glutamate with either 10 mM lactate (low ATP) or 10 mM pyruvate (high ATP)). The tubules were then incubated in Krebs-Henseleit saline using two different phosphate concentrations and the same substrate mixture. The ATP content in tubular cells was modified by these treatments, ranging from 2.2 to 5.7 mM. The oxygen uptake by the tubules was measured before and after application of a small amount of nystatin (0.05 mM, 6 mumol/g wet wt.), added to impose an identical and submaximal increment of work to the Na(+)-K+ ATPase in tubules, irrespective of their ATP condition. This manoeuvre was followed by the addition of 1 mM ouabain to inhibit the sodium pump and quantify the respiration related to the activity of the Na+ pump. No significant effect of the ATP content on the respiratory cost of the Na(+)-K+ ATPase activity was noted when the [ATP] was above the normal concentration of approximately 3.0 mM before or after introduction of nystatin. In a second group of experiments, tubules were treated with 0.1 mM digitonin (13 mumol/g wet wt.) and resuspended in intracellular-like and sodium-free medium. The respiration was measured before and after the addition of increasing Mg-ATP concentrations (0-12 mM). A fixed quantity of Na+ (20 mM) was then introduced before ouabain was applied. The oxygen uptake was measured in these three conditions. We observed a fixed increment of ouabain-sensitive respiration upon stimulation of the pump activity by sodium at ATP concentrations ranging from 2 to 7 mM. The same observation applied when the free energy released from ATP hydrolysis ranged from -50 to -56 kJ.mol-1 and when the [ATP]/[ADP].[Pi] ratio ranged from 1.5 to 7.5 mM-1. These results suggest that the Na+:ATP stoichiometry of the Na(+)-K+ ATPase is not modified by [ATP] in dog cortical tubules when the ATP content is at or above the physiological value. Furthermore, the stoichiometry of the pump does not appear to change when the phosphate potential and (or) the free energy released from ATP hydrolysis are altered.

Effects of anoxia on cellular damage in the incubated mouse diaphragm

Satisfactory ultrastructural integrity of the mouse diaphragm was maintained in vitro in modified Krebs-Henseleit saline for 3h when the rate of oxygenation was 2.8 ml sec −1 (95% O 2 + 5% CO 2). Hypoxic (O 2 = 1.6 – 2.0 ml sec −1) or anoxic (95% N 2 + 5% CO 2) conditions triggered typical Ca-triggered myofilament damage, believed to be induced by a rise in [Ca] i. It was unaffected by omission of Ca from the saline, but the muscle was protected at 7.8 °C. ‘High-O 2’ gassing (10 ml sec −1) also caused a characteristic, but different, damage with swollen sarcoplasmic reticulum and spacing of the myofibrils.

Improvement of renal preservation by verapamil with 24-hour cold perfusion in the isolated rat kidney.

There is substantial evidence that increased cellular calcium may activate processes that lead to cellular injury and death, and calcium entry blockers (CEB) have been shown to protect against renal ischemic injury. This approach has been used experimentally to enhance kidney preservation during both warm and cold ischemia. In the present study, the effect of the CEB verapamil on kidney function after 24 hr of hypothermic (4-7 degrees C) perfusion was examined and compared with simple cold storage with Eurocollins' solution (4 hr), 4 or 24 hr cold perfusion, without the addition of verapamil. The cold perfusion media consisted of 3% albumin in phosphate-free Krebs-Henseleit saline supplemented with 5 mM glucose. Cold perfusion was performed at 40 mmHg perfusion pressure with either 0 (C) or 5 microM verapamil (V) added to the cold perfusion media. Renal functional parameters of plasma flow (RPF), inulin clearance (Cin), fractional (FRNa+) and net sodium reabsorption (TNa+) were assessed during 60 min of reperfusion at 37 degrees C using 6.7% albumin in Krebs-Henseleit saline supplemented with glucose, inulin, and 20 amino acids. There was no increase in RPF with V (33 +/- 1 vs. 32 +/- 2 ml/min/g,NS) but Cin was significantly higher (271 +/- 30 vs. 168 +/- 20 microliter/min/g P less than 0.01) with V. Preservation of tubular function by V was demonstrated by an increase in FRNa+ (84 +/- 5 vs. 57 +/- 8%, P less than .01), TNa+ (32 +/- 6 vs. 15 +/- 3 mumol/min/g, P less than .01) and renal adenosine triphosphate (ATP) concentration (8.0 +/- 5 vs. 4.7 +/- 1.0 mumol/g dry tissue, P less than .01). Thus, V appears not only to enhance kidney preservation with warm and cold ischemia but also improves renal function, as assessed by glomerular filtration rate (GFR) tubular function, and tissue ATP concentration with 24-hr cold perfusion.

Studies on the acetylating capacity of human colonic epithelial cells for 5-aminosalicylic acid.

Following oral administration of sulphasalazine, 5-aminosalicylic acid (5-ASA) is present in serum at very low concentrations and is mostly in the acetylated form. Analysis of rectal mucosal biopsies from patients on sulphasalazine maintenance therapy has shown that the predominant metabolite present is N-acetyl ASA (Clin Sci 1987: 72; 79P). The authors have now investigated whether this acetylation takes place in the epithelium. Isolated colonic epithelial cell suspensions were prepared in Krebs-Henseleit saline from fresh human resection specimens by EDTA digestion and mechanical disaggregation. Cell suspensions remained viable, as determined by lactate production, over 3 hours. Aliquots of cell suspension were incubated with 5-ASA, 0.1 mM, and glucose, 5 mM, at 37°C for 1 or 2 hours. Following incubation, the supernatant was analysed by high performance liquid chromatography for the presence of acetyl-ASA. Following washing, the cell pellet was disrupted ultrasonically, and intracellular acetyl-ASA was also measured. Production of acetyl-ASA in 1 hour was 112.5 (+27 SD) nmol/g dry weight (extracellular) and 48 (+15.6 SD) nmol/g dry weight (intracellular). No intracellular 5-ASA was detected, suggesting rapid and complete acetylation. A cytosolic fraction of the epithelial cells was prepared by centrifugation of homogenized cells at 13,000 g. In the presence of 5-ASA and acetyl-CoA, this enzyme solution produced acetyl-ASA linearly at a rate of 37 nmol/minutes/ mg protein. No acetyltransferase activity was found in the brush border. The cytosol was further purified by passing it through a Superose 12 gel filtration column. Maximum acetyltransferase activity was found in the fraction corresponding to a molecular weight of approximately 30,000. Thus, the human colonic epithelial cell is probably the primary site of acetylation of 5-ASA, and the N-acetyltransferase activity is found in the cytosol.

Advantages of perfluorochemical perfusion in the isolated working rabbit heart preparation using 31P-NMR

Quantitative 31P-NMR and enzymatic analysis of high-energy phosphates were used to characterize an isolated perfused working rabbit heart preparation. In this model, the left side of the heart works against a physiological after-load. Two perfusates, Krebs-Henseleit saline and the perfluorocarbon emulsion FC-43 (perfluorotributylamine), were evaluated in their ability to maintain cardiac function and high-energy phosphate metabolites over a period of 2–3 h. Adenine nucleotides ATP, ADP, phosphocreatine and inorganic phosphate (P i) were measured by 31P-NMR while monitoring cardiac output and coronary flow. Intracellular pH was determined using the chemical shift of P i. At the end of each experiment, hearts were freeze clamped and enzymatically assayed for adenine nucleotides, phosphocreatine and P i. In every experiment, hearts perfused with FC-43 emulsion maintained the same rate of cardiac output as hearts perfused with Krebs-Henseleit saline, but with half the coronary flow rate: FC-43, 22 ± 2.5 ( n = 5), Krebs-Henseleit saline 42 ± 2.7 ( n = 6) ml/min, P < 0.001. Hearts perfused with FC-43 emulsion showed higher [phosphocreatine] and [ATP] measured by 31P-NMR. For [phosphocreatine]: FC-43 3.2 ± 0.7 ( n = 5), Krebs-Henseleit saline 1.7 ± 0.2 ( n = 6) μmol/g wet wt., P < 0.01. For [ATP]: FC-43 1.8 ± 0.7 ( n = 5), Krebs-Henseleit saline 0.9 ± 0.2 ( n = 6) μmol/g wet wt., P < 0.02. [phosphocreatine] and [ATP] determined by 31P-NMR values were identical within experimental error to those values obtained by enzymatic analysis. Comparing [P i] determined by both methods, 36% of P i in FC-43-perfused hearts, and only 24% of P i in Krebs-Henseleit saline-perfused hearts were visible by NMR, indicating that a large proportion of P i is bound in the intact functioning heart. Similar results were obtained for [ADP]. Using the combined techniques of 31P-NMR and enzymatic assay, we have shown in this model of the isolated working rabbit heart preparation, that FC-43 emulsion maintains significantly better function and high-energy phosphate levels than Krebs-Henseleit saline.

Enhanced reabsorption of bicarbonate and phosphorus by the addition of amino acids in the isolated perfused rat kidney

In the isolated perfused kidney, a defect in urinary acidification has been noted [1]. Nevertheless, this preparation which used rat kidney with albumin saline solution as perfusate including glucose as substrate has also been used for the study of distal acidification, because of urinary acidification reached in the extreme metabolic acidosis [2, 3]. Recently, in a model of the isolated perfused rat kidney, it was demonstrated clearly that adding amino acids results in the improvement and stability of renal function which are associated with the amelioration of histological damage of the thick ascending limb of Henle [4]. This observation prompted us to re-evaluate acid-base parameters of this preparation with respect to the effect of amino acids. Our results show that the addition of amino acids mixture leads to significant increase and stability of the reabsorption of HCO3 and phosphorus as well as the constant urine pH. Methods. Kidneys from male Sprague-Dawley rats weighing 340 to 400 g were perfused in vitro as previously described [2, 5] except for using 18-gauge metal needles instead of glass cannulas. Calculated mean artery pressure was maintained at 90 mm Hg during the experiments. Perfusion was carried out for 85 mm with the last 60 mm divided into four clearance periods with 6.7% bovine albumin Krebs-Henseleit saline solution, equilibrated with 97% 02/5% CO2. The perfusion medium contained 5 mM glucose with (N = 5), or without amino acids (N = 5), the composition of which was similar to that of a previous study [4]. Prior to each experiment 2 ml of concentrated essential amino acid solution (M. A. Bioproducts, Walkersville, Maryland, USA) were added to 100 ml of a prepared perfusate. The amino acid solution contained 12 amino acids including cystine (2.4 mg) and nine other amino acids individually. This procedure provides the similar composition of amino acids as previously described [4] except for the presence of cystine. Perfusate pH was adjusted to about 7.40. Urine and perfusate samples were obtained at 15-mm intervals; urine samples were collected under mineral oil. Analysis. Pco2 and pH of perfusate and urine were measured by a blood gas system (BMS3-MK2 , Radiometer, Copenhagen, Denmark). Sodium and potassium were measured with a flame photometer, and '4C-inulin was used for the determination of

Binding of branched-chain 2-oxo acids to bovine serum albumin.

1. Binding of branched-chain 2-oxo acids to defatted bovine serum albumin was shown by gel chromatography and equilibrium dialysis. 2. Equilibrium-dialysis data suggest a two-side model for binding in Krebs-Henseleit saline at 37 degrees C with n1 = 1 and n2 = 5. Site association constants were: 4-methyl-2-oxovalerate, k1 = 8.7 x 10(3) M-1, k2 = 0.09 x 10(3) M-1; 3-methyl-2-oxovalerate, k1 = 9.8 x 10(3) M-1, k2 = 0.08 x 10(3) M-1; 3-methyl-2-oxobutyrate, k1 = 1.27 x 10(3) M-1, k2 = less than 0.05 x 10(3) M-1. 3. Binding of 4-methyl-2-oxovalerate to defatted albumin in a phosphate-buffered saline, pH 7.4, gave the following thermodynamic parameters: primary site delta H0(1) = -28.6kJ . mol-1 and delta S0(1) = -15.2J . mol-1 . K-1 (delta G0(1) = -24.0kJ . mol-1 at 37 degrees C) and secondary sites delta H0(2) = -25.4kJ . mol-1 and delta S0(2) = -46.1J . mol-1 . K-1 (delta G0(1) = -11.2kJ . mol-1 at 37 degrees C). Thus binding at both sites is temperature-dependent and increases with decreasing temperature. 4. Inhibition studies suggest that 4-methyl-2-oxovalerate may associate with defatted albumin at a binding site for medium-chain fatty acids. 5. Binding of the 2-oxo acids in bovine, rat and human plasma follows a similar pattern to binding to defatted albumin. The proportion bound in bovine and human plasma is much higher than in rat plasma. 6. Binding to plasma protein, and not active transport, explains the high concentration of branched-chain 2-oxo acids leaving rat skeletal muscle relative to the concentration within the tissue, but does not explain the 2-oxo acid concentration gradient between plasma and liver.

Paracetamol metabolism by the isolated perfused rat kidney

Renal metabolism of drugs and their effects on tubular function depend on the entry of the drug into specific renal cells. In turn, this depends on differential regional blood flow and the relative rates of delivery of the drug to luminal and contraluminal surfaces of the cell following filtration at the glomerulus. In studies of renal pharmacology, it is desirable to have an intact kidney with a clearly defined experimental system. Such conditions are provided in part by the isolated perfused rat kidney, in which a buffered, oxygenated medium containing only bovine serum albumin in Krebs-Henseleit saline is perfused into the renal artery. Glomerular filtration, fractional sodium reabsorption, and the composition of the recirculated perfusate, as well as the urine and the kidney itself, can all be analyzed. Bowman [1] has recently outlined the perfusion methods relevant to pharmacology. This paper briefly reviews the approaches to renal drug handling and drug effects using the isolated perfused kidney. Its main purpose is to report detailed studies with tritiated paracetamol demonstrating direct renal involvement in metabolism of this drug and to offer possible mechanisms for the development of analgesic nephropathy and nephrotoxicity of other drugs.

Method of preparing isolated colonic epithelial cells (colonocytes) for metabolic studies.

Suspensions of isolated colonic epithelial cells (colonocytes) have been obtained from rats by incubating everted lengths of colon with EDTA at 37 degrees C in Krebs-Henseleit saline from which calcium was omitted and containing 0.25% (w/v) bovine serum albumin. Measurements of oxygen consumption and lactate production by cell suspensions indicate that they are metabolically active for at least one hour. The method has been modified for the preparation of isolated epithelial cells from the human colon by including an enzyme digestion step and by increasing the concentration of EDTA to 10 mM. Human colonocytes have been obtained either from normal mucosa (ascending and descending colon) or from the mucosa in ulcerative colitis (descending colon). Oxygen consumption of human cell suspensions is lower than in the rat but in colonocytes from both species the rate is increased by glucose and by n-butyrate, a normal constituent of the colonic lumen. The method yields metabolically active cell suspensions from diseased colonic mucosa and promises to be of value for biochemical studies of ulcerative colitis.

Inhibition of hepatic gluconeogenesis by dichloroacetate

Gluconeogenesis from lactate by isolated hepatocytes suspended in a low bicarbonate medium is effectively inhibited by the hypoglycemic agent dichloroacetate. With this medium dichloroacetate suppresses the accumulation of the components of the malateaspartate shuttle, limits mitochondrial utilization of cytoplasmic reducing equivalents, and makes the availability of pyruvate and/or oxaloacetate limiting for gluconeogenesis. Much less inhibition is observed with hepatocytes suspended in a medium (Krebs−Henseleit saline) containing physiological concentrations of bicarbonate. No inhibition is observed with Krebs-Henseleit saline supplemented with lysine as a source of amino groups for the malate-aspartate shuttle. Thus, dichloroacetate inhibition of gluconeogenesis is observed only when hepatocytes are incubated in a medium deficient in bicarbonate and amino acids. This means that the action of dichloroacetate as a hypoglycemi agent is best explained by stimulation of peripheral tissue utilization of glucose and potential precursors for hepati gluconeogenesis rather than by direct inhibition of hepatic gluconeogenesis.