Presence and function of ß-adrenergic receptors in primary equine bronchial epithelia cells.

The ß-adrenergic receptor (ß-AR) plays an important role in regulating a variety of cell and organ functions in different animal species and is an important target in asthma pathogenesis and therapy. The ß-AR expression and function in equine bronchial epithelial cells (EBEC) were not known but innervation and significant decrease in receptor level were reported in the equine bronchial tissues from asthmatic horses. 125I-iodocyanopindolol (ICYP) binding studies were undertaken in primary freshly isolated and cultured EBEC to identify the presence of the ß-ARs. The receptor distribution was assessed using subtype-selective ß-AR antagonists (ICI 118 551 (ß2) and CGP 20712A (ß1). The ß-AR function was confirmed by measuring the agonist-induced intracellular cAMP accumulation in freshly isolated and cultured EBEC. In both freshly isolated and cultured EBEC, the specific ICYP binding was saturable and of high affinity. The maximal receptor density (Bmax) was 9763 ± 140 binding sites/cell (mean ± SEM, n = 7) and 10575 ± 194 binding sites/cell (mean ± SEM, n = 5) in freshly isolated and cultured EBEC, respectively. The receptor affinity to the ligand (KD) was also not different between the two cell conditions. ICI 118.551 displaced ICYP with 25 000-fold higher affinity than CGP 20712A. Moreover, in both fresh isolated and cultured EBEC, cAMP-accumulation was stimulated with a rank-order of potency of isoproterenol > adrenaline > noradrenaline. These results highlight the ß2-AR to be a key subtype in both freshly isolated and cultured primary EBEC.

Distribution and properties of cardiac and pulmonary ß-adrenergic receptors in corn snakes (Pantherophis guttatus) and Boa constrictor (Boa constrictor).

The aim of the present study was to characterize ß-adrenergic receptors in the snake heart and lung of corn and Boa constrictor snakes. The ß-adrenergic receptor binding sites were studied in purified heart and lung membranes using the specific ß-adrenergic receptor antagonist [125J]-iodocyanopindolol (ICYP) and subtypes using selective ß1-adrenergic receptor antagonist CGP-20712A and selective ß2-adrenergic receptor antagonist ICI-118.551. A saturable and specific ß-adrenergic receptor binding site was detected in cardiac membranes with maximal receptor density (Bmax) of 43.99 ±â€¯3.86 fmol/mg protein (corn snake) and 58.07 ±â€¯2.88 fmol/mg protein (Boa constrictor) as well as KD of 24.21 ±â€¯7.38 pM (corn snake) and 21.48 ±â€¯3.85 pM (Boa constrictor) and in lung membranes (Bmax fmol/mg protein: 55.95 ±â€¯16.28 (corn snake) and 107.00 ±â€¯14.21 (Boa constrictor); KD pM: 71.25 ±â€¯21.92 (corn snake) and 55.04 ±â€¯18.68 (Boa constrictor)). Competition-binding studies showed ß-adrenergic receptors with low affinities to the ß2-selective adrenergic receptor antagonist and high affinity binding to ß1-selective adrenergic receptor antagonist in both heart and lung tissues of both snake species, suggesting the presence of high population of the post-synaptic ß1-adrenergic receptor subtype. It seems that the presence of the predominant ß1-subtype also in lung tissues may indicate the importance of the vascular system in the snake lung.

Release kinetics of tumor necrosis factor-α and interleukin-1 receptor antagonist in the equine whole blood.

BACKGROUND: Horses are much predisposed and susceptible to excessive and acute inflammatory responses that cause the recruitment and stimulation of polymorphnuclear granulocytes (PMN) together with peripheral blood mononuclear cells (PBMC) and the release of cytokines. The aim of the study is to develop easy, quick, cheap and reproducible methods for measuring tumor necrosis factor alpha (TNF-α) and interleukin-1 receptor antagonist (IL-1Ra) in the equine whole blood cultures ex-vivo time- and concentration-dependently. RESULTS: Horse whole blood diluted to 10, 20 and 50 % was stimulated with lipopolysaccharide (LPS), PCPwL (a combination of phytohemagglutinin E, concanavalin A and pokeweed mitogen) or equine recombinant TNF-α (erTNF-α). TNF-α and IL-1Ra were analyzed in culture supernatants, which were collected at different time points using specific enzyme-linked immunosorbent assays (ELISA). Both cytokines could be detected optimal in stimulated 20 % whole blood cultures. TNF-α and IL-1Ra releases were time-dependent but the kinetic was different between them. PCPwL-induced TNF-α and IL-1Ra release was enhanced continuously over 24-48 h, respectively. Similarly, LPS-stimulated TNF-α was at maximum at time points between 8-12 h and started to decrease thereafter, whereas IL-1Ra peaked later between 12-24 h and rather continued to accumulate over 48 h. The equine recombinant TNF-α could induce also the IL-1Ra release. CONCLUSIONS: Our results demonstrate that similar to PCPwL, LPS stimulated TNF-α and IL-1Ra production time-dependently in whole blood cultures, suggesting the suitability of whole blood cultures to assess the release of a variety of cytokines in health and diseases of horse.

Agonist-induced ß2-adrenoceptor desensitization and downregulation enhance pro-inflammatory cytokine release in human bronchial epithelial cells.

It is not clear whether increased asthma severity associated with long-term use of ß2-adrenoceptor (ß2-AR) agonists can be attributed to receptor degradation and increased inflammation. We investigated the cross-talk between ß-AR agonist-mediated effects on ß2-AR function and expression and cytokine release in human bronchial epithelial cells. In 16HBE14o(-) cells grown in the presence and absence of ß-AR agonists and/or antagonists, the ß2-AR density was assessed by radioligand binding; the receptor protein and mRNA was determined using laser scanning cytometer and RT-PCR; cAMP generation, the cytokines IL-6 and IL-8 release were determined using AlphaScreen Assay and ELISA, respectively. Isoprenaline (ISO) and salbutamol (Salbu) induced a concentration- and time-dependent significant decrease in ß2-AR density. Both Salbu and ISO reduced cAMP generation in a concentration-dependent manner while in same cell culture the IL-6 and IL-8 release was significantly enhanced. These effects were antagonized to a greater extent by ICI 118.551 than by propranolol, but CGP 20712A had no effect. Reduction of the ß2-AR protein and mRNA could be seen when cells were treated with ISO for 24 h. Our findings indicate a direct link between cytokine release and altered ß2-AR expression and function in airway epithelial cells. ß2-AR desensitization and downregulation induced by long-term treatment with ß2-AR agonists during asthma may account for adverse reactions also due to enhanced release of pro-inflammatory mediators and should, thus, be considered in asthma therapy.

Comparative study of the effects of fetal bovine serum versus horse serum on growth and differentiation of primary equine bronchial fibroblasts.

BACKGROUND: Airway fibroblasts have become a critical addition to all facets of structural lung tissue changes such as in human asthma and chronic obstructive pulmonary disease, but little is known about their role in the equine recurrent airway obstruction, a disease that resembles to the human asthma. Since the equine bronchial fibroblasts (EBF) have not been isolated and characterized yet, the use of defined medium was investigated. RESULTS: Primary EBF were cultured on non-collagen coated flasks without serum or in the presence of fetal bovine serum (FBS) or horse serum (HS) or in serum depleted medium. EBF cultured in serum-free basal media and those serum deprived were not able to proliferate and even exhibited considerable cell death. In media containing FBS or HS, proliferation of the cells was reproducible between different primary cultures and cells demonstrated expression of vimentin. Large variations were found in the ability of FBS and HS to support growth and differentiation of EBF in monolayer culture. Indications of growth-promoting actions, increasing passage number as well as maintaining fibroblast morphology were found rather in FBS than in HS. EBF culturing in HS needed longer doubling and confluence time. The protein content of the cell pellets was higher in EBF cultured in medium containing HS than FBS. Alpha-smooth muscle actin seemed to be less expressed in EBF cultured in medium containing FBS than those in HS. CONCLUSIONS: In sum, serum addition to basal EBF medium enhanced EBF differentiation into myofibroblasts, and these findings are useful to develop in vitro fibroblast culture models that mimic in vivo physiological processes and to study airway disease mechanisms and remodeling.

Assessing the effect of breed, age, and sex on muscarinic receptor distribution in atria and ventricles of turkeys.

Atrial and ventricular myocardium from young (6-wk-old), young adult (3-6-mo-old), and aged (14-15-mo-old) meat-type (B.U.T. Big 6) and wild-type (Cröllwitzer) turkeys were used to study the influence of age and sex on cholinergic muscarinic receptors using [3H]-N-methyl-scopolamine (3H-NMS) binding studies. In both breeds, saturation experiments indicated the presence of regional-, sex-, and age-related differences in the density of cholinergic muscarinic receptors (Bmax), that is, a decrease or increase. Except for right atria, Bmax was decreased in both male and female B.U.T. Big 6 hearts with increasing age. Similarly, a negative correlation between Bmax and age could be seen in female and male atria of Cröllwitzer turkeys, while positive correlation could be seen in right and left ventricles of male, and only right ventricles of female Cröllwitzer turkeys. The affinity of the receptor (KD) was not affected by age, sex and breed. In all cardiac chamber tissues, the M2-subtype was shown to be predominant followed by the M3-subtype and to a lesser extent the M1-subtype. Aspects of this age-dependent remodeling of the heart differ between sexes, resulting in maladaptive changes in older turkeys with a high degree of frailty. These observations may help explain why males and females are susceptible to different cardiovascular diseases as they age and why frail older adults are most often affected by these diseases.

Dimethyl sulfoxide favors the emetic efficacy of lycorine in beagle dogs - a novel strategy for the treatment of poisoning.

Poisoning in small animals represents an ongoing hazard and therapeutic problem in veterinary medicine. Therapeutic induction of emesis in time enables a fast elimination of a toxic compound resulting in a shortened course of poisoning and a higher safety level thereafter, which decisively improves prognosis and treatment. Lycorine is a reliable emetic drug in beagle dogs without serious side effects thought to be more beneficial in tolerability and efficacy than the rarely used apomorphine. Therefore, this study investigates efficacy and tolerability of differently composed potential drug formulations of lycorine hydrochloride for s.c. administration in dogs as an emetic principle. By emesis response analysis four dimethyl sulfoxide (DMSO)-based active pharmaceutical ingredient (API) formulations were favored. Two of them (F5 and F6) qualified for further drug development. Both formulations ensure a safe pharmacologically induced emesis within about 30 min after injection, suitable for use as an in time decontaminant in acute poisoning of dogs. DMSO-based formulations were well tolerated and offer a novel promising strategy for treatment of poisoning.

The presumed atypical chemokine receptor CXCR7 signals through G(i/o) proteins in primary rodent astrocytes and human glioma cells.

SDF-1/CXCL12 binds to the chemokine receptors, CXCR4 and CXCR7, and controls cell proliferation and migration during development, tumorigenesis, and inflammatory processes. It is currently assumed that CXCR7 would represent an atypical or scavenger chemokine receptor which modulates the function of CXCR4. Contrasting this view, we demonstrated recently that CXCR7 actively mediates SDF-1 signaling in primary astrocytes. Here, we provide evidence that CXCR7 affects astrocytic cell signaling and function through pertussis toxin-sensitive G(i/o) proteins. SDF-1-dependent activation of G(i/o) proteins and subsequent increases in intracellular Ca(2+) concentration persisted in primary rodent astrocytes with depleted expression of CXCR4, but were abolished in astrocytes with depleted expression of CXCR7. Moreover, CXCR7-mediated effects of SDF-1 on Erk and Akt signaling as well as on astrocytic proliferation and migration were all sensitive to pertussis toxin. Likewise, pertussis toxin abolished SDF-1-induced activation of Erk and Akt in CXCR7-only expressing human glioma cell lines. Finally, consistent with a ligand-biased function of CXCR7 in astrocytes, the alternate CXCR7 ligand, I-TAC/CXCL11, activated Erk and Akt through ß-arrestin. The demonstration that SDF-1-bound CXCR7 activates G(i/o) proteins in astrocytes could help to explain some discrepancies previously observed for the function of CXCR4 and CXCR7 in other cell types.

Expression and functional coupling of liver ß2 - adrenoceptors in the human hepatocellular carcinoma.

AIM: Hepatocellular carcinoma (HCC) is one of the most common cancers and a leading cause of death worldwide. There are now multiple lines of evidence demonstrating that the ß-adrenoceptor ( ß-AR) signaling plays an important role in the progression and metastasis of cancer and may become a novel target for cancer therapy. Little information exists regarding the status of ß-ARs and their postreceptor intracellular signaling cascade in the development of human HCC. This study was conducted to detect the expression signal transduction of the ß-ARs in liver membranes obtained from patients with HCC and elucidate their possible implication on HCC development. METHODS: The ß-AR density and subtype distribution were determined by receptor binding studies. Protein levels of the ß(2)-AR and G(s)(α) protein were determined by Western blot analysis. The receptor coupling efficiency and biochemical activities of the adenylate cyclase(AC) was also determined. RESULTS: In HCC liver membranes, the ß(2)-AR density was higher than the density in the nonadjacent nontumor liver membranes. The ß(2)-AR protein expression was 1.5-fold increased as compared with nonmalignant controls, and positively correlated with the receptor density. The G s protein expression as well as the receptor, AC and G protein-stimulated activation of the cAMP formation was reduced in HCC. CONCLUSION: The ß(2)-AR was upregulated in human HCC. Despite this upregulation of the receptor,there was an altered postreceptor signal transduction in HCC liver. The mechanisms responsible for this change in the growth of HCC and the nature of this alteration remain unclear.

Identification and characterization of ß-adrenergic receptors in isolated primary equine tracheal epithelial cells.

Responses and functions of airway epithelial cells are stimulated by ß2-agonists via the ß2-adrenergic receptors (ß2-ARs)-G(s)-protein-cAMP-system, thus, affecting airway inflammation such as in asthma and equine recurrent airway obstruction (RAO). Though horses can be used as large animal model for human asthma, evaluation of the expression and functions of the ß-AR system in primary equine airway epithelial cells has not been yet carried out. Thus, for the first time, we determined the ß-AR density and subtype distribution by [¹²5I]-iodocyanopindolol (ICYP) binding, examined ß-AR function by cAMP assay as well as their expression by western blot analysis and immunocytochemical staining in primary equine tracheal epithelial cells (ETEC). Cells were collected from 19 horses and cultured subsequently. The specific ICYP binding was saturable and of high affinity: in freshly isolated cells the receptor density (B(max)) and ICYP affinity (K(D)) for ß-ARs were 12727 ± 883 binding sites/cell and 31.78 ± 6.57 pM, respectively, and in cultured ETEC 3730 ± 212 binding sites/cell and 15.26 ± 3.37 pM, respectively. The ß-AR subtype assessed by ß1-selective (CGP 20712A) and ß2-selective (ICI 118.551) adrenergic receptor antagonists demonstrated that the ß2-AR subtype predominated (>95%) in both cell populations (p < 0.001). The ß-AR agonists increased cAMP formation with a rank order of potency: isoproterenol > epinephrine > norepinephrine. ICI 118.551 (100 nM) significantly blocked (p < 0.05) isoproterenol-induced cAMP accumulation but not CGP 20712A (300 nM). Western blot analyses and immunocytochemical staining further indicated the expression of the ß(2)-AR subtype in both cell preparations. Our data indicate that in acutely dissociated and primary cultured ETEC the ß(2)-AR-AC system is expressed, but varies considerably between the two preparations.

Increased antioxidant capacity in the plasma of dogs after a single oral dosage of tocotrienols.

The intestinal absorption of tocotrienols (TCT) in dogs is, to our knowledge, so far unknown. Adult Beagle dogs (n 8) were administered a single oral dosage of a TCT-rich fraction (TRF; 40 mg/kg body weight) containing 32 % α-TCT, 2 % ß-TCT, 27 % γ-TCT, 14 % δ-TCT and 25 % α-tocopherol (α-TCP). Blood was sampled at baseline (fasted), 1, 2, 3, 4, 5, 6, 8 and 12 h after supplementation. Plasma and chylomicron concentrations of TCT and α-TCP were measured at each time point. Plasma TAG were measured enzymatically, and plasma antioxidant capacity was assessed by the Trolox equivalent antioxidant capacity assay. In fasted dogs, levels of TCT were 0·07 (sd 0.03) µmol/l. Following the administration of the TRF, total plasma TCT peaked at 2 h (7.16 (SD 3.88) µmol/l; P < 0.01) and remained above baseline levels (0.67 (SD 0.44) µmol/l; P < 0.01) at 12 h. The TCT response in chylomicrons paralleled the increase in TCT in plasma with a maximum peak (3.49 (SD 2.06) µmol/l; P < 0.01) at 2 h post-dosage. α-TCP was the major vitamin E detected in plasma and unaffected by TRF supplementation. The Trolox equivalent values increased from 2 h (776 (SD 51.2) µmol/l) to a maximum at 12 h (1130 (SD 7.72) µmol/l; P < 0.01). The results show that TCT are detected in postprandial plasma of dogs. The increase in antioxidant capacity suggests a potential beneficial role of TCT supplementation in the prevention or treatment of several diseases in dogs.

Growth and differentiation of primary and passaged equine bronchial epithelial cells under conventional and air-liquid-interface culture conditions.

BACKGROUND: Horses develop recurrent airway obstruction (RAO) that resembles human bronchial asthma. Differentiated primary equine bronchial epithelial cells (EBEC) in culture that closely mimic the airway cells in vivo would be useful to investigate the contribution of bronchial epithelium in inflammation of airway diseases. However, because isolation and characterization of EBEC cultures has been limited, we modified and optimized techniques of generating and culturing EBECs from healthy horses to mimic in vivo conditions. RESULTS: Large numbers of EBEC were obtained by trypsin digestion and successfully grown for up to 2 passages with or without serum. However, serum or ultroser G proved to be essential for EBEC differentiation on membrane inserts at ALI. A pseudo-stratified muco-ciliary epithelium with basal cells was observed at differentiation. Further, transepithelial resistance (TEER) was more consistent and higher in P1 cultures compared to P0 cultures while ciliation was delayed in P1 cultures. CONCLUSIONS: This study provides an efficient method for obtaining a high-yield of EBECs and for generating highly differentiated cultures. These EBEC cultures can be used to study the formation of tight junction or to identify epithelial-derived inflammatory factors that contribute to lung diseases such as asthma.

In vivo assessment of antiemetic drugs and mechanism of lycorine-induced nausea and emesis.

Lycorine is the main alkaloid of many Amaryllidaceae and known to cause poisoning with still unknown mechanisms. Longer lasting toxicological core symptoms of nausea and emesis may become a burden for human and animal patients and may result in substantial loss of water and electrolytes. To optimise the only empirical symptomatic antiemetic drug treatment at present, it is important to elucidate the causative involved targets of lycorine-induced emesis. Therefore, in the current study, we have tested the actions of a various antiemetic drugs with selective receptor affinities on lycorine-induced nausea and emesis in vivo in dogs. Beagle dogs were pre-treated in a saline vehicle-controlled crossover and random design with diphenhydramine, maropitant, metoclopramide, ondansetron or scopolamine prior lycorine administration (2 mg/kg subcutaneously). In vivo effects were assessed by a scoring system for nausea and emesis as well as by the number and lag time of emetic events for at least 3 h. Moreover, plasma pharmacokinetic analysis was carried out for ondansetron before and after lycorine injection. The data show that histaminergic (H1), muscarinic and dopaminergic (D2) receptors are presumably not involved in lycorine-induced emetic effects. While ondansetron significantly reduced the number of emetic events, lycorine-induced emesis was completely blocked by maropitant. Only ondansetron also significantly decreased the level of nausea and was able to prolong the lag time until onset of emesis suggesting a preferential participation of 5-HT3 receptors in lycorine-induced nausea. Thus, it is the first in vivo report evidencing that predominantly neurokinin-1 (NK1) and to a lesser extent 5-hydroxytryptamine 3 (5-HT3) receptors are involved in lycorine-induced emesis facilitating a target-oriented therapy.

The Rho kinase (ROCK) inhibitor Y-27632 reduces the ß2-adrenoceptor density but enhance cAMP formation in primary equine bronchial epithelial cells.

The present study addresses the effect of the Rho-kinase (ROCK) inhibitor Y-27632 on the ß2-adrenoceptor density and ß-agonist-stimulated intracellular second messenger cAMP formation in primary equine bronchial epithelial cells (EBEC). Y-27632 significantly decreased the ß2-adrenoceptor number (Bmax) without markedly affecting the receptor affinity (dissociation constant, KD) to the radioligand [125I]-iodocyanopindolol (ICYP). In contrast, Y-27632 augmented the ß-agonist-stimulated intracellular cAMP production. Herein, Y-27632 markedly increased the maximal cAMP responses (Emax) (isoproterenol > epinephrine > norepinephrine) but did not shift the ß-agonist concentration-effect curves to the left. The ß2-selective antagonist ICI 118.551 and the ß1/ß2-antagonsit propranolol but not the ß1-selctive antagonist CGP 20712A reversed the isoproterenol-induced cAMP formation equally in Y-27632-treated and control EBEC, suggesting the effect was merely related to the ß2-subtype. These results show that Y-27632 differentially regulates the receptor density and function. Thus, these findings provide the first evidence that the functional interaction of the ß2-adrenoceptor and Rho-kinase (ROCK) signaling pathways decreases the receptor expression but enhances receptor downstream cAMP formation. This differential regulation of the receptor density and function by Y-27632 should be further reconsidered with regard to the beneficial effect of the drug in asthma therapy.

Expression of muscarinic acetylcholine receptors in turkey cardiac chambers.

The aim of the present study was to characterize the specific binding sites for [N-methyl-3H]-scopolamine ([3H]-NMS), a radioligand for labeling muscarinic acetylcholine receptors (mAChRs), in membranes of four heart chambers obtained from adult male British United Turkey (BUT) Big 6 ("meat-type") and Cröllwitzer ("wild-type") turkeys. MAChR subtypes were examined by inhibiting [3H]-NMS binding with subtype selective non-labelled receptor antagonists. In all left and right atria as well as left and right ventricles of both turkey breeds, the specific [3H]-NMS binding was saturable, reversible and of high affinity (KD range: 0.5-1.0 nM). The maximum receptor density (Bmax) was not significantly different between the four cardiac chambers of BUT Big 6 turkeys, but a significant difference was found between atria and ventricles of Cröllwitzer turkeys. Moreover, significant lower Bmax was found in the atria of Cröllwitzer turkeys than in the atria of BUT Big 6, while the ventricular Bmax was significantly higher. In all cardiac chambers, unlabeled mAChR antagonists competed for specific [3H]-NMS binding sites in a concentration-dependent manner, suggesting the presence of the M3 and M2 receptor subtypes, whereby the latter was the predominant subtype. The presence of the M1 subtype could not be excluded. In conclusion, there was a difference between BUT Big 6 ("meat-type") and Cröllwitzer ("wild-type") turkeys with regard to receptor density in heart chambers with dominant M2 and M3 receptor subtypes.

Oral bioavailability of quercetin from different quercetin glycosides in dogs.

Although the flavonol quercetin is used as a supplement in commercial dog food, data on quercetin bioavailability in dogs are not available. Thus, we investigated quercetin bioavailability (measured as area under the concentration-time curve) in nine adult beagle dogs at an oral dose of 10 mg/kg body weight (b.w.). The major fraction (>80 %) of flavonols circulating in blood plasma were conjugated metabolites of quercetin. The absolute bioavailability of quercetin (i.e. the fraction that reaches the systemic circulation) was only about 4 %. We also compared the oral bioavailability between the aglycone quercetin and its more often used glucorhamnoside (rutin) and 3-O-glucoside (isoquercitrin) at an equimolar dose of 30 mumol/kg b.w. (corresponding to 10 mg quercetin/kg). Quercetin and isoquercitrin were mainly absorbed in the small intestine with isoquercitrin being one and a half times more bioavailable than quercetin. Maximal plasma concentration after isoquercitrin treatment was 0.89 (sem 0.07) mumol/l. Although quercetin absorption from rutin was delayed, relative bioavailability was not lower than from the aglycone itself. The latter observation is in clear contrast to findings in human subjects, pigs or rats and might indicate that rutin is a better source of quercetin in dogs than in other species. However, potential in vivo quercetin effects beyond the gastrointestinal tract are limited by the intensive metabolism as well as by the rather low bioavailability of this flavonol.

Effects of Hypoxia and Acidosis on Cardiac Electrophysiology and Hemodynamics. Is NHE-Inhibition by Cariporide Still Advantageous?

Hypoxia often leads to severe cardiac malfunctions. It is assumed that intracellular calcium overload is -inter alia- responsible for left ventricular (LV) deterioration. Inhibition of the sodium-proton exchanger (NHE), which finally inhibits/slows calcium overload, may ameliorate cardiac function. Our aim was to evaluate cariporide, an inhibitor of NHE1 in a Langendorff-perfused heart model. To discriminate a potentially different impact of extracellular acidosis and hypoxia we examined 48 Chinchilla Bastard rabbits divided into 8 experimental groups: control group (pH = 7.4, O2 = 100%) without or with cariporide (1 µM), acidosis group (pH = 7.0, O2 = 100%) without or with cariporide (1 µM), hypoxia group (pH = 7.4, O2 = 40%) without or with cariporide (1 µM) and hypoxia+acidosis group (pH = 7.0, O2 = 40%) without or with cariporide (1 µM). Hearts were subjected to acidotic/hypoxic conditions for 90 min followed by 60 min of reperfusion. Hypoxia and hypoxia+acidosis led to a severe deterioration of LV function with a decrease in LV pressure by about 70% and an increase of end-diastolic pressure from 6.7 ± 0.6 to 36.8 ± 5.4 (hypoxia) or from 7.0 ± 0.2 to 18.6 ± 4.1 (hypoxia+acidosis). Moreover, maximum contraction velocity decreased from about 1,800 mmHg/s to 600 mmHg/s during hypoxia ± acidosis and maximum relaxation velocity deteriorated from -1,500 mmHg/s to about -600 mmHg/s. During reperfusion hearts subjected to hypoxia+acidosis recovered faster than hearts subjected to hypoxia alone, reaching control levels after 5 min of reperfusion. Electrophysiologic analysis revealed an 1.2 fold increase in both dispersion of activation-recovery interval and in total activation time in the hypoxia ± acidosis group. Cariporide application significantly improved LV hemodynamics and electrophysiology in the hypoxia group but not in the group subjected to hypoxia+acidosis. Immunohistologic analysis of cardiac specimen revealed a significant increase of factors involved in hypoxia/reperfusion injury like nitrotyrosine and poly-ADP-ribose as well as apoptosis-inducing factors like AIF or cleaved-caspase 3 in LV after hypoxia ± acidosis. ATP was reduced by hypoxia but not by acidosis. Again, cariporide mitigated these processes only in the hypoxia alone group, but not in the group with additional acidosis. Acidosis without hypoxia only marginally disturbed LV function and electrophysiology, and was not affected by cariporide. Thus, our study demonstrated that several detrimental effects of hypoxia were mitigated or abrogated by acidosis and that NHE-inhibition improved only hypoxia-induced cardiac dysfunction.

Segment-dependent activation of muscarinic acetylcholine receptor-mediated [35S]Guanosine-5'-O-(gamma-thiotriphosphate) binding in airway tissue membranes.

Muscarinic acetylcholine receptor (mAChR)-mediated guanine nucleotide-binding regulatory protein (G protein) activation and the functional interaction between receptors and the respective G proteins were investigated using an agonist-induced [(35)S]guanosine-5'-O-(gamma-thiotriphosphate) ([(35)S]GTPgammaS)-binding approach in membranes of 3 native equine airway segments (trachea, bronchus and lung), which differ tremendously in mAChR density and subtype distribution; especially subtypes that couple negatively to adenylyl cyclase through G(i/0) proteins, i.e. M(2) receptors. The assay was initially optimized by determining the influence of incubation time, guanosine 5'-diphosphate (GDP), MgCl(2) and NaCl on basal and agonist-stimulated [(35)S]GTPgammaS binding. In standard assays, the presence of 10 mumol/l GDP, 10 mmol/l MgCl(2) and 200 mmol/l NaCl increased carbachol-induced specific [(35)S]GTPgammaS binding in a segment- and receptor-density-dependent manner. Hereby, mAChR agonists stimulated [(35)S]GTPgammaS binding with a rank order of potency (oxotremorine M > carbachol > acetylcholine), and in a specific segment- and receptor-density-dependent manner (trachea > bronchus > lung). The increase in the specific [(35)S]GTPgammaS binding was potently inhibited by the mAChR antagonist atropine. Pertussis toxin and N-ethylmaleimide as G(i/0) protein ADP-ribosylating and alkylating agents reduced basal and agonist-stimulated [(35)S]GTPgammaS binding. The mAChR-stimulated [(35)S]GTPgammaS binding serves as a useful method for investigating the functional interaction between mAChRs and their respective G proteins in native airway tissue membranes of equines.

Modulation of TNF-α, IL-1Ra and IFN-γ in equine whole blood culture by glucocorticoids.

Glucocorticoids are important drugs in the treatment of many inflammatory, autoimmune and allergic diseases in humans and animals. We investigated the effects of hydrocortisone and dexamethasone on TNF-α, IL-1Ra and INF-γ release in stimulated whole blood cell culture from healthy horses. Whole blood cell cultures proved to be useful for the characterization of the anti-inflammatory properties of new drugs. Diluted equine whole blood was exposed to lipopolysaccharide (LPS) and PCPwL (a cocktail consisting of phythemagglutinin E, concanavalin A, pokeweed mitogen and lipopolysaccharide) in the presence or absence of hydrocortisone and dexamethasone (10-12 - 10-5 M). TNF-α and IL-1Ra (LPS) as well as IFN-γ (PCPwL) levels were measured in the supernatants using specific enzyme-linked immunosorbent assay (ELISA). The LPS-induced TNF-α and IL-1Ra as well as the PCPwL-induced IFN-γ levels were more potently suppressed by dexamethasone than by hydrocortisone in a concentration-dependent manner. Dexamethasone inhibited TNF-α, IL-1Ra and IFN-γ with the half maximal inhibition concentration (IC50) values of 0.09 µM, 0.453 µM and 0.001 µM, respectively, whereas hydrocortisone inhibited these cytokines with lower IC50 values of 1.45 µM, 2.96 µM and 0.09 µM, respectively. Our results suggest that the equine whole blood test system is useful and reliable to evaluate drug effects and immunological alterations and offers several advantages including simple and cheap performance in physiological and pathological conditions.