Minimally-invasive catheterization of the portal, hepatic and cranial mesenteric veins and the abdominal aorta for quantitative determination of hepatic metabolism in dairy cows.

The objective of this study was to establish a minimally-invasive, ultrasound (US)-guided technique for the placement of indwelling catheters into the portal, hepatic, and cranial mesenteric veins as well as the abdominal aorta. Catheters were placed in eight healthy dairy cows on day 1. The patency of catheters was tested daily until day 14 when a necropsy was carried out. On day 6, energy intake and hepatic net output of glucose, removal of lactate, and oxygen were determined in seven cows. Post mortem examination revealed that all implanted catheters were in the intended locations. Loss of patency in one portal vein catheter on day 9 was attributable to a fibrin clot. Significant correlations were found between mean energy intake and mean hepatic plasma flow (r=0.91; P=0.004), hepatic glucose output (r=0.81; P=0.027) and hepatic removal of lactate (r=-0.70; P=0.08) and oxygen (r=-0.77; P=0.039), as well as between hepatic glucose net output and removal of lactate (r=-0.92; P=0.004). Minimally-invasive, US-guided transcutaneous catheter placement into the cranial mesenteric, portal and hepatic veins as well as the technique for catheterization of the abdominal aorta appear to be safe, and suitable for studies of quantitative hepatic metabolism in cattle.

Lectin histochemistry of the temporal gland of the African elephant (Loxodonta africana).

The study demonstrates the free sugar spectrum of the secretion of the tubuloaveolar temporal gland of the African elephant (Loxodonta africana), using lectin histochemistry. In the elephant, the spectrum contained, besides strongly varying amounts of α-D-mannose, very remarkable reactions for α-D-galactose and to a certain extent also for α-D-N-acetyl-galactosamine or a/ß-D-N-acetylglucosamine. This is in contrast to the free sugar spectrum of the secretion of the mammalian apocrine tubular skin glands. Considering also that the production of any water binding mucus seems to be negligible, the variations of the free sugar contents found probably originate from another important task of the secretory cells of the temporal gland. This means that our findings corroborate the view of highly active acquired immunity by an intensive processing and presenting of lipid antigens by dendritic cells (APC), in particular of the CD1 family.

Technical note: Analysis of total lipid and triacylglycerol content in small liver biopsy samples in cattle.

A procedure is described for analyzing total lipid (TL) and triacylglycerol (TAG) in 2 sequential steps using small amounts (<100 mg) of bovine liver tissue. The TL was measured gravimetrically and TAG was measured enzymatically in the TL extract, using an automated analyzer. For gravimetric TL determination in milligrams per gram of liver fresh weight (FW), TL was extracted from homogenized tissue samples with hexane:isopropanol (at 20 degrees C, 24 h, constant agitation). The routine method was modified by adding a second hexane extraction step to optimize lipid extraction. The dry lipid extract was dissolved in hexane and aliquoted according to TL content for TAG analysis. An extra incubation period of 16 h was included for complete hydrolysis of TAG, using microbial lipase and nonaethylene glycol monododecyl ether detergent, before TAG was measured enzymatically using commercial test kits. Triolein was used as an internal standard. Repeated TL analysis (n = 3) of liver specimens from 10 cows (range, 40 to 314 mg/g of FW) yielded a mean CV of 2.2%, whereas repeated TAG analysis (range, 4 to 260 mg/g of FW) yielded a mean intraday CV of 2.5% (n = 5) and a mean interday CV of 3.4% (n = 4). Intraday (n = 5) and interday (n = 4) CV for repeated TAG analysis in triolein standards were <1 and <3%, respectively. Recovery of TAG in triolein standards varied between 99 and 103%. In part 2 of the experiment, hepatic TL and TAG were measured in 150 German Holstein cows to verify the test method in a large sample size. For repeated hepatic TL (n = 3) and TAG (n = 5) determination, mean CV of <2.8 and <1.5%, respectively, were found. The proportion of TAG relative to TL increased linearly to a breakpoint of approximately 100 mg TL/g of FW, at which point it reached a plateau at approximately 68%, indicating an accumulation of other lipid fractions in hepatic tissue with hepatic TL above the breakpoint. Calculation of hepatic TAG from TL was reasonably accurate when a 2-slope linear broken-line model (r(2) = 0.98) was used. Above a TL of approximately 40 mg/g of FW, calculated TAG values deviated by only +/-15% from measured hepatic TAG.

Demonstration of lipids in plastic resin-embedded sections of skin material.

Two different fluorescence stains, green: 5-hexadecanoylaminofluorescein, and red: BODIPY(R) 665/676 [(E,E)-3, 5-bis-(4-phenyl-1,3-butadienyl)-4,4-difluoro-4-bora-3a, 4a-diaza-s-indacene, produced good results regarding the demonstration of glycolipids, free fatty acids and triglycerides in mammalian skin material that had been embedded in a water miscible plastic resin (Technovit(R) 7100). In this way, functional aspects of specific structures (epidermal barrier region, sebaceous glands) could be characterized histochemically in the integument of five mammalian species with sparse or dense hair coats.

The effects of dietary phosphorus deficiency on surface pH and membrane composition of the mucosa epithelium in caprine jejunum.

In ruminants, the uptake of inorganic phosphate (P(i)) across the intestinal mucosa epithelium by Na-dependent and Na-independent mechanisms is a main regulatory factor in P homeostasis. The aim of the study was to elucidate to which extent Na-independent mechanisms, including pH effects or composition of mucosal brush-border membranes, could be involved in positive stimulation of P(i) absorptive processes seen under the P deficient condition. Therefore, luminal, surface and intracellular pH of the jejunal epithelial cells in control and P depleted goats were compared and biochemical analyses of membrane phospholipids in the apical membrane of the jejunal epithelium were performed. Dietary P depletion resulted in decreased plasma P(i) levels. While pH in jejunal ingesta was not significantly changed, P depletion resulted in a significantly lower surface pH in the crypt region compared to control animals (7.62 +/- 0.02 vs. 7.77 +/- 0.04, n = 4, P < 0.01). Inhibition of apical Na(+)/H(+)-exchange resulted in an increase of the jejunal surface pH in P depleted animals by 0.07 +/- 0.01 (n = 6, P < 0.01) and 0.05 +/- 0.01 (n = 6, P < 0.01) for the villus and the crypt region, respectively. This increase were inversely correlated with the initial surface pH prior to inhibition. In contrast to surface pH, intracellular pH of the jejunal epithelium and the phospholipid composition of the apical jejunal membrane were not affected by P depletion. Although the data suggest the existence of a Na(+)/H(+)-exchange mechanism at the luminal surface of goat jejunum they do not support the hypothesis that adaptational processes of active P(i) absorption from goat jejunum in response to low dietary P could be based on "non P(i) transporter events".

Effect of chloride on pH microclimate and electrogenic Na+ absorption across the rumen epithelium of goat and sheep.

Active Na+ absorption across rumen epithelium comprises Na+/H+ exchange and a nonselective cation conductance (NSCC). Luminal chloride is able to stimulate Na+ absorption, which has been attributed to an interaction between Cl-/HCO3- and Na+/H+ exchangers. However, isolated rumen epithelial cells also express a Cl- conductance. We investigated whether Cl- has an additional effect on electrogenic Na+ absorption via NSCC. NSCC was estimated from short-circuit current (Isc) across epithelia of goat and sheep rumen in Ussing chambers. Epithelial surface pH (pHs) was measured with 5-N-hexadecanoyl-aminofluorescence. Membrane potentials were measured with microelelectrodes. Luminal, but not serosal, Cl- stimulated the Ca2+ and Mg2+ sensitive Isc. This effect was independent of the replacing anion (gluconate or acetate) and of the presence of bicarbonate. The mean pHs of rumen epithelium amounted to 7.47 +/- 0.03 in a low-Cl- solution. It was increased by 0.21 pH units when luminal Cl- was increased from 10 to 68 mM. Increasing mucosal pH from 7.5 to 8.0 also increased the Ca2+ and Mg2+ sensitive Isc and transepithelial conductance and reduced the fractional resistance of the apical membrane. Luminal Cl- depolarized the apical membrane of rumen epithelium. 5-Nitro-2-(3-phenylpropylamino)-benzoate reduced the divalent cation sensitive Isc, but only in low-Cl- solutions. The results show that luminal Cl- can increase the microclimate pH via apical Cl-/HCO3- or Cl-/OH- exchangers. Electrogenic Na+ absorption via NSCC increases with pH, explaining part of the Cl- effects on Na+ absorption. The data further show that the Cl- conductance of rumen epithelium must be located at the basolateral membrane.

Carbon dioxide differentially affects the cytokine release of macrophage subpopulations exclusively via alteration of extracellular pH.

BACKGROUND: The improved outcome after endoscopic surgery has been attributed to less surgical trauma. However, the underlying mechanisms are not fully understood, and direct effects of CO2 used for pneumoperitoneum, cellular acidification, and/or the lack of air contamination have been postulated to additionally modulate immune functions during endoscopic surgery. We investigated the effects of CO2 incubation, extracellular acidification, and air contamination on the inflammatory response of two distinct macrophage populations. METHODS: R2 and NR 8383 rat macrophage cell lines were used. Interleukin-6 (IL-6) and nitric oxide after lipopolysaccharide (LPS) stimulation were determined in these sets of experiments: incubation in 100% CO2, 5% CO2, and room air for 2h; incubation at pH 7.4, 6.5, and 5.5 for 2 h in 5% CO2; and incubation in 100% CO2, 5% CO2 and room air in fixed pH 6.3. The extracellular pH was monitored during incubation. We determined the alteration of intracellular pH in cells subjected to extracellular acidification by fluorescence microscopy. RESULTS: Extracellular pH decreased to 6.3 during 100% CO2 incubation. IL-6 release was reduced after CO2 incubation in NR 8383 cells and increased in R2 cells (p < 0.05). It was not altered by air incubation. Decreasing the extracellular pH to 6.5 mimicked the effects of CO2 and a decrease to 5.5 suppressed IL-6 release in both cell lines. In fixed pH at 6.3, CO2 and air incubation had no effect. CO2 and pH had no impact on nitric oxide release and vitality. Intracellular pH decreased with extracellular acidification without significant difference between the two cell lines. CONCLUSIONS: A decrease in extracellular pH during incubation in CO2 differentially affects IL-6 release in macrophage subpopulations. This may explain contradictory results in the literature. Moreover, we demonstrated that air contamination does not affect macrophage cytokine release. The decrease in extracellular pH is the primary underlying mechanism of the alteration of macrophage cytokine release after CO2 incubation, and it appears that the ability to maintain intracellular pH is not determined by the effects of CO2 or extracellular acidification.

Comparative distribution of sulphur, thiols and disulphides in the porcine stratum corneum.

Biochemical, histochemical and cytochemical analyses were used to determine the sulphur contents and the thiol and disulphide distribution in the stratum corneum (SC) of the wild boar (WB), a large domestic pig breed (DP) and the Goettingen miniature pig (GMP). The sulphur contents (% DW) were different in the three animal types (WB: 1.70-1.38 body, 0.54 ear; DP: 0.84-0.53 body, 0.50 ear; GMP: 2.28-2.51 body, 2.66 ear). The results of the histochemical analysis of SH- and -S-S- groups were clear, and densitometrical extinctions were highest in most body regions of the GMP for thiols and disulphides, followed by the DP for thiols, and the WB for disulphides. Absolute SC thickness was highest in the body of the GMP (62-80 mum), and generally lowest in the ear (20-38 mum) of all animal types. Relative SC thickness was the same for all animals in the body (40-66%), but lower in the ear (30%). Only -S-S- concentrations were correlated with SC thickness, and primarily in the GMP. Cytochemical analysis showed that high sulphur concentrations were obvious particularly in the CCE of corneal cells in the DP, as compared to the cytoplasm. Intracellular sulphur distribution was homogenous in the WB, and in the GMP, although in the latter at a higher concentration level. The results indicate breed-related effects on keratinisation in porcine corneal cells. Only the SC of the outer side of the ear of DP females is recommended as a model for humans.

[Effects of excess caloric fat feeding on the lipid metabolism in Shetland ponies]. / Auswirkungen überkalorischer Fettfütterung auf den Fettstoffwechsel bei Shetland Ponys.

To investigate the influence of overweight and dietary fat supplementation on lipid and insulin glucose metabolism of Shetland ponies, eight Shetland pony geldings were fed a hypercaloric (30 MJ DE/150 kg bwt. and day) fat diet (10% fat as soybean oil) or a carbohydrate control diet for nine months until ponies gained an overweight of 15%. Afterwards oral glucose tolerance tests (oGTT; 5, 6 mmol/kg bwt.) were performed after a 12 hour fast and after a fast which led to an increase of plasma triglyceride concentrations to a threshold of 3 mmol/l (36-65 hrs.). Plasma concentrations of glucose, insulin, triglycerides and non esterified fatty acids (NEFA) were determined for 480 minutes after the glucose load. Ponys having had received the control diet tended to a higher insulin secretion in case of both oGTTs, whereas the glucose tolerance was similar in both groups but lower than in ponies of normal weight. During the oGTTs after fasting leading to the plasma triglyceride threshold, triglyceride concentrations decreased significantly (p < 0.05) faster and stronger in fat fed ponies. Additionally, fat fed pony showed significantly (p < 0.05) lower NEFA levels. The results of this study demonstrate a positive effect of fat feeding on the triglyceride clearance of overweight Shetland ponies.

Apoptosis induction in vivo and fate of apoptotic material in the colon of the guinea pig.

Short-chain fatty acids (SCFA) in particular butyrate are regarded as an energy source acting in beneficial, protective manner on the colonic mucosa. Previous investigations showed that the colonic mucosa bathed in Ussing chamber with a solution lacking butyrate induced massive apoptosis of epithelial cells. The apoptotic material (bodies and cells) was shed at the mucosa surface. In the present study we aimed to investigate the effects caused in vivo on the colonic mucosa by the absence of butyrate. For this purpose the colon of guinea pigs was perfused in situ with solutions either containing or lacking butyrate. The results show that within 2h of perfusion without butyrate a large amount of epithelial cells underwent apoptosis as in the in vitro experiments. However, apoptotic material instead to be extruded at the epithelial surface accumulates into the intercellular spaces from which it becomes removed by an unusual high number of macrophages. These, engorged with phagocytozed material, lie assembled in a layer below the epithelium. Similar alterations have not been observed after perfusion in the presence of butyrate. The results suggest that this SCFA may protect the colonic mucosa in that it prevents apoptosis. The alterations occurring during 2h of its absence allow to assume that a protracted butyrate deprivation may lead to a breakdown of the integrity of the mucosa thus influencing differently the activity of the macrophages.

Helicobacter pylori augments the acid inhibitory effect of omeprazole on parietal cells and gastric H(+)/K(+)-ATPase.

BACKGROUND: In duodenal ulcer patients, intragastric acidity during omeprazole treatment is significantly lower before Helicobacter pylori eradication than after cure. AIMS: To determine if H pylori enhances the acid inhibitory potency of omeprazole in isolated parietal cells and on H(+)/K(+)-ATPase. METHODS: Rat parietal cells and pig gastric membrane vesicles enriched in H(+)/K(+)-ATPase activity were incubated with H pylori and the H pylori fatty acid cis 9,10-methyleneoctadecanoic acid (MOA), and the inhibitory effects of omeprazole on parietal cell acid production, H(+)/K(+)-ATPase enzyme activity, and ATPase mediated proton transport were assessed. RESULTS: In isolated parietal cells, H pylori and MOA increased the acid inhibitory potency of omeprazole 1.8 fold. H pylori did not affect the inhibitory potency of omeprazole on H(+)/K(+)-ATPase enzyme activity. In proton transport studies, H pylori (intact bacteria and sonicate) and MOA accelerated the onset of the inhibitory effect of omeprazole and enhanced the proton dissipation rate in response to omeprazole. H. pylori itself increased proton permeability at the vesicle membrane. CONCLUSION: Our results show that H pylori augments the acid inhibitory potency of omeprazole in parietal cells and enhances omeprazole induced proton efflux rate from gastric membrane vesicles. We suggest that omeprazole unmasks the permanent effect of H pylori on proton permeability at the apical parietal cell membrane, which is counteracted in the absence of a proton pump inhibitor by a reserve H(+)/K(+)-ATPase capacity.

Residual chloride secretion in intestinal tissue of deltaF508 homozygous twins and siblings with cystic fibrosis. The European CF Twin and Sibling Study Consortium.

BACKGROUND & AIMS: Cholinergic stimulation of chloride secretion is impaired in the intestines of patients with cystic fibrosis (CF). However, intestinal chloride secretion has been observed in patients with mild CF mutations. The aim of this study was to investigate residual Cl(-) secretion in the intestine of DeltaF508 homozygous CF patients, and examine the contribution of cystic fibrosis transmembrane conductance regulator (CFTR) and alternative Cl(-) conductances. Twins and siblings with identical CFTR genotypes were investigated to determine the impact of factors other than CFTR on chloride secretion. METHODS: Chloride secretion in rectal tissue was investigated by applying Ca(2+) and adenosine 3',5'-cyclic monophosphate (cAMP)-linked agonists before and after the inhibition of alternative Cl(-) conductances with 4,4'-diisothiocyanostilbene-2, 2'-disulfonic acid (DIDS). RESULTS: cAMP-mediated Cl(-) secretion was observed in 73% of patients, and 20% showed DIDS-sensitive Ca(2+)-activated Cl(-) secretion. This DIDS-sensitive alternative chloride conductance was seen only in CF patients who also responded to cAMP agonists. Chloride secretion was more concordant within monozygous twins than within dizygous pairs. CONCLUSIONS: These results suggest the presence of CFTR-mediated Cl(-) secretion in a subgroup of patients, implying that a portion of deltaF508 CFTR can be processed in vivo and function as a chloride channel in the apical membrane of intestinal cells. Moreover, a considerable number of deltaF508 homozygous patients express chloride conductances other than CFTR in their intestinal epithelia.

Cytokine signaling through Stat3 activates integrins, promotes adhesion, and induces growth arrest in the myeloid cell line 32D.

Hematopoietic cell development and function is dependent on cytokines and on intercellular interactions with the microenvironment. Although the intracellular signaling pathways stimulated by cytokine receptors are well described, little is known about the mechanisms through which these pathways modulate hematopoietic cell adhesion events in the microenvironment. Here we show that cytokine-activated Stat3 stimulates the expression and function of cell surface adhesion molecules in the myeloid progenitor cell line 32D. We generated an erythropoietin receptor (EpoR) isoform (ER343/401-S3) that activates Stat3 rather than Stat5 by substituting the Stat3 binding/activation sequence motif from gp130 for the sequences surrounding tyrosines 343 and 401 in the receptor cytoplasmic region. Activation of Stat3 leads to homotypic cell aggregation, increased expression of intercellular adhesion molecule 1 (ICAM-1), CD18, and CD11b, and activation of signaling through CD18-containing integrins. Unlike the wild type EpoR, ER343/401-S3 is unable to support long term Epo-dependent proliferation in 32D cells. Instead, Epo-treated ER343/401-S3 cells undergo G(1) arrest and express elevated levels of the cyclin-dependent kinase inhibitor p27(Kip1). Sustained activation of Stat3 in these cells is required for their altered morphology and growth properties since constitutive SOCS3 expression abrogates homotypic cell aggregation, signaling through CD18-containing integrins, G(1) arrest, and accumulation of p27(Kip1). Collectively, our results demonstrate that cytokine-activated Stat3 stimulates the expression and function of cell surface adhesion molecules, indicating that a role for Stat3 is to regulate intercellular contacts in myeloid cells.

Erythropoietin receptors that signal through Stat5 or Stat3 support fetal liver and adult erythropoiesis: lack of specificity of stat signals during red blood cell development.

Erythropoietin (Epo) is essential for formation of mature red blood cells (RBC). However, the function of Epo receptor (EpoR)-dependent signaling pathways in the regulation of erythropoiesis remains unclear. To determine whether specific Stat signals are required for RBC development, we changed the Stat signaling specificity of the EpoR. The wild-type EpoR activates only Stat5. Thus, we substituted the major Stat5 binding sites (residues 343 and 401) in the EpoR cytoplasmic region with the Stat3 binding/activation motif from gp130. We demonstrated that activated EpoRs containing a single substitution stimulate Stat5 and Stat3, whereas an EpoR with both substitutions stimulates Stat3 but not Stat5. We then determined the ability of these receptors to support fetal liver and adult erythropoiesis. Our results show that erythropoiesis is stimulated by EpoRs that activate Stat5, both Stat5 and Stat3, or Stat3 in place of Stat5. These findings demonstrate that the specificity of EpoR Stat signaling is not essential for RBC development.

Surface pH at the basolateral membrane of the caecal mucosa of guinea pig.

Since the major mechanisms responsible for regulation of intracellular pH of enterocytes are located in the basolateral membrane, respective effects may be expected on pH in the compartment near the basolateral membrane. A method was established to estimate the pH at the basolateral membrane (pHb) of isolated caecal epithelia of guinea pig using pH-sensitive fluorescein attached to lectin (lens culinaris). In the presence of bicarbonate and a perfusion solution-pH of 7.4, pHb was 7.70 +/- 0.15. In the absence of bicarbonate or chloride as well as by inhibition of the basolateral Cl--HCO-3 exchange with H2-DIDS, pHb was reduced near to solution-pH. Inhibition of the basolateral Na+-H+ exchanger by adding a sodium- and bicarbonate-free, low-buffered solution increased pHb. Decrease of pH of serosal perfusion solution to 6.4 provoked a similar decrease of pHb to solution pH. Short-chain fatty acids (SCFA) added to the mucosal solution caused a slight decrease of pHb. SCFA added to the serosal side alkalized pHb. However, in the presence of bicarbonate pHb returned quickly to the initial pHb, and after removal of SCFA a transient acidification of pHb was seen. These responses could not be inhibited by MIA or H2-DIDS. We conclude that no constant pH-microclimate exists at the basolateral side. The regulation of the intracellular pH of enterocytes reflects pHb. The slightly alkaline pHb is due to the bicarbonate efflux. Data support the presence of an SCFA--HCO-3 exchange.

Maintenance and regulation of the pH microclimate at the luminal surface of the distal colon of guinea-pig.

1. The fluorescent dye 5-N-hexadecanoyl-aminofluorescein (HAF) was used to study the mechanisms involved in maintaining a relatively constant luminal surface pH (pHs) in the distal colon of the guinea-pig. The fatty acyl chain of the HAF molecule inserts into the apical membrane of epithelial cells. This allows a continuous measurement of the surface pH for several hours. 2. The localization of HAF was confirmed by confocal laser-scanning microscopy and by using monoclonal antibodies against fluorescein. The insertion of HAF into the apical membrane of the colonocytes did not change the transepithelial conductance or the short-circuit current of the epithelium. 3. With the HAF method a pH microclimate was confirmed at the colonic surface. Although the pH of the bulk luminal solution was decreased in bicarbonate-containing solution from 7.4 to 6.4 the pHs changed only in the range 7.54-6.98. 4. In the absence of bicarbonate pHs almost followed changes of bulk luminal pH. In the presence of bicarbonate there was a decrease in pHs after removal of chloride from the luminal side and an increase in pHs after addition of butyrate to the luminal solution. This suggests the involvement of a bicarbonate-anion exchange in bicarbonate secretion: a Cl--HCO3- as well as a short-chain fatty acid--HCO3- exchange. 5. The apical K+-H+-ATPase in the distal colon of guinea-pig has little influence on pHs in the presence of physiological buffer concentrations. 6. Our findings indicate that bicarbonate plays a major role in maintaining the pH microclimate at the colonic surface.

Isolation and lipid composition of apical and basolateral membranes of colonic segments of guinea Pig.

In adapting several methods of membrane isolation we established a successful way to purify apical and basolateral membranes of guinea pig colon in a parallel procedure. The conventional purification control by marker enzymes was applied. In addition, luminal membrane proteins were stained with Texas Red. Apical and basolateral enterocyte membranes were enriched 10- to 12-fold by differential precipitation and via a continuous sorbitol gradient. The membrane fractions were examined with regard to their phospholipid (PL) and fatty acid patterns and to their cholesterol content. Fluorescence polarization studies were carried out using 1,6-diphenyl-1,3, 5-hexatrien. Remarkable differences in the fatty acid pattern of the proximal and the distal colon were seen. Due to a higher content of oleic acid the saturation index of the apical membranes of the proximal colon is lower compared to that of the apical membranes of the distal colon (0.34 +/- 0.03 vs 0.42 +/- 0.05). The cholesterol content of the apical membranes of the proximal colon is markedly higher than that of the apical membranes of the distal colon (3.42 +/- 0.14 vs 1.88 +/- 0.29 mol/mol PL). There are no differences in the fluidity of these apical membranes. We assume a balancing mechanism between the cholesterol content and the amount of saturated PL-fatty acids.

Role of short-chain fatty acids in the hind gut.

Short-chain fatty acids (SCFA) are produced by microbial fermentation in the hindgut in considerable amounts. Most of the anions in hindgut contents are SCFA, mainly acetate, propionate and butyrate. SCFA are rapidly absorbed. Mechanisms involved in the transepithelial transport are discussed. Besides the contribution to the overall energy metabolism of animals or men, SCFA have a number of further important effects on the colonic mucosa. Factors affecting the pH of compartments in the mucosa, cell swelling, stimulation of mucin release and of mucosal blood flow are mentioned. Controversial reports are known on the role of SCFA in the metabolism of colonocytes. In spite of the conflicting opinions on the interaction between SCFA metabolism and the development of colitis ulcerosa, diverticulosis and colorectal cancer seems to exist. The obscure differences between the effects of SCFA on cell proliferation, differentiation and apoptosis of colonocytes in vivo and in vitro indicate that besides direct effects of SCFA systemic effects such as neural and humoral factors are of crucial importance. The opposing effects of SCFA on proliferation and apoptosis in normal colonocytes and in colonic cancer cells may open possibilities for prevention and/or therapy of patients with colonic diseases.

Studies on equine lipid metabolism. 1. A fluorometric method for the measurement of lipolytic activity in isolated adipocytes of rats and horses.

A simple and sensitive method for direct and continuous monitoring of free fatty acid (FFA) release, by measuring the pH-sensitive change in relative fluorescence intensity of seminaphthofluorescein (SNAFL-1) is described. The method was designed to use a small number of adipocytes isolated from fat pads of rats and biopsy specimens of horses for the detection of decreasing pH in fat cell suspensions caused by released FFA into the incubation medium. Species specific differences of lipolysis were demonstrated when adipocytes of rats and horses are incubated with stimulators or inhibitors of lipolysis. Norepinephrine (NE) stimulated lipolysis in fat cells of rats whereas adipocytes of horses showed a measurable release of FFA when concomitantly incubated with NE and adenosine deaminase (ADA) or NE and 8-Phenyltheophylline (8-PT), respectively). The incubation of equine fat cells with NE and ADA did not influence the antilipolytic response to insulin. The method described enables micro-scaled in vitro studies on lipolytic activity.

Effect of SCFA on intracellular pH and intracellular pH regulation of guinea-pig caecal and colonic enterocytes and of HT29-19a monolayers.

The response of the intracellular pH (pHi, measured with BCECF) of the caecal and distal colonic epithelium of guinea pig and of monolayers of HT29 clone 19a cells on the addition of short-chain fatty acids (SCFA) was assessed. Addition of SCFA to the luminal side of these cells had no major effect on pHi, independent of whether the apical Na+/H+ exchange or the apical K+/H+ ATPase was inhibited or not. Addition of SCFA to the serosal side, on the other hand, caused a marked decrease of pHi, followed by an effective regulation back to basal values, and after removal of the acid, the cells became alkalinized. Intracellular pH is mainly regulated by mechanisms in the basolateral membrane. The basolateral Na+/H+ exchanger and the Cl-/HCO3- exchanger were mainly responsible for pHi regulation. Inhibition studies are consistent with a NHE-1 type Na+/H+ exchanger in the basolateral membranes. The apical Na+/H+ exchanger of caecal enterocytes and in HT29 cells, and the apical K+/H+ ATPase in the apical membrane of the distal colon have no or little influence on pHi regulation. The comparison shows that the HT29-19a cell line is an adequate model for studying pHi phenomena of hind gut epithelial cells.