J. Physiol. (1975), 245, pp. 333-350 With 3 text-ftgure8 Printed in Great Britain

333

ACIDIFICATION IN THE RAT PROXIMAL JEJUNUM

BY J. A. BLAIR, M. L. LUCAS AND A. J. MATTY From the Departments of Chemistry and Biological Sciences, University of Aston in Birmingham, Birmingham B4 7ET

(Received 10 April 1974) SUMMARY

1. Production of hydrogen ions by the rat proximal jejunum was investigated using the everted sac technique. 2. Acidification occurred in the absence of glucose, increasing on addition of glucose to reach a maximal value. An apparent Km of 1-78 mM was derived for the glucose-dependent process. 3. Acidification in the presence of glucose was inhibited by 10 mM-2: 4dinitrophenol, 10 mm phlorrhizin, 10 mm aminophylline and anaerobiosis. 4. Histamine, ethylenediamine tetraacetic acid (EDTA), ouabain and acetazolamide, compounds known to alter acid production in gastric mucosa had no effect on jejunal acidification. 5. Galactose and 3-0-methylglucose failed to increase acidification; in contrast, mannose and fructose did lead to increases, indicating metabolic origin of the hydrogen ions. 6. Serosal and mucosal lactate production were measured and the calculated percentage of hydrogen ions possibly derived from this source was shown to account for only a small proportion of acidification. 7. The greatest increase in acidification with minimal simultaneous production of lactate occurred with ATP which was shown not to enter intestinal tissues. 8. A hypothesis for acidification, that of the break-down at the mucosal surface of ATP from intracellular metabolic sources, is proposed and its relevance to the postulated microclimate is discussed. INTRODUCTION

Many types of tissue, notably frog skin (Fleming, 1957; Emilio, Machado & Menano, 1970), turtle bladder (Steinmetz, 1967; Brodsky & Schilb, 1967) and various mammalian gastric tissues, acidify the medium in which they are incubated. This phenomenon has also been studied in human, dog and rat jejunum (Klotz & Schloerb, 1971; Robinson, 1935; Wilson, 1953, 1954; Wilson & Kazyak, 1957). Some workers (Schanker, Tocco, Brodie &

J. A. BLAIR AND OTHERS 334 Hogben, 1958; Hogben, Tocco, Brodie & Schanker, 1959) have proposed the existence of a microclimate of hydrogen ions next to the jejunal wall at a concentration far higher than that in the bulk phase of the lumen. Also, a model for the transport of folic acid has been proposed incorporating the microclimate hypothesis (Smith, Matty & Blair, 1970a; Blair, Johnson & Matty, 1974). The microclimate has never been demonstrated directly in situs in the intestine although Caldwell (1958) showed a similar phenomenon in crab muscle fibre. A microclimate would require a source of hydrogen ions to maintain the lower pH and acidification might reflect diffusion of hydrogen ions from the microclimate into the bulk phase. The purpose underlying the present experiments was to investigate the cause of acidification in rat proximal jejunum and to relate the findings to the pH microclimate hypothesis. More substance might be lent to this hypothesis if it were shown that acidification is an event occurring at the mucosal surface. Data are presented supporting this view. METHODS

Source of chemicals. Histamine, adenosine triphosphate, 3-O-methylglucose and acetazolamide (2-acetylamino-1:3:4-thiadiazole-5-sulphonamide) were obtained from Koch-Light Ltd, Colnbrook, U.K.; dinitrophenol and ethylenediamine tetraacetic acid disodium salt (EDTA) from Hopkin & Williams Ltd, Essex, U.K.; aminophylline, imidazol and ouabain from Sigma Chemical Co., U.S.A.; galactose from Fisons Ltd, Loughborough, U.K.; and lactate estimation kits from Boehringer & Soehne, G.M.B.H., Mannheim, Germany. Pure U-14C ATP of known specific activity was obtained from the Radiochemical Centre, Amersham, Bucks. Preparation of everted sacs. The experimental technique was the everted sac technique with two different procedures depending on the buffer used. Male Wistar rats (200-250 g) were starved for 48 hours with access to water only. Initially, rats were killed by a blow on the head and the jejunum removed. To alleviate the anoxic effects caused by intense vasoconstriction due to any shock on handling (Robinson, Antonioli & Mirkovitch, 1966), in later experiments, rats were anaesthetized with ether prior to removal of the jejunum. Although ether caused a moderate reduction (Fig. 1) in the phenomenon under investigation, the jejunum had a less blanched appearance and the practice was continued. However, all statistical comparisons were made between animals similarly treated. The technique of preparing the sacs was that originally described by Wilson & Wiseman (1954) and in greater detail by Wiseman (1961). Since a number of sacs were taken from each rat, sacs were made in sequence and treatments were allocated to sacs after randomization by a latin square design. Six 3 5 cm long everted sacs were made, filled with 0-2 ml. fluid and maintained at 4°C until all the sacs were made. The viability of these preparations was checked by measuring sac respiration, glucose and water transport and the transmural potential difference. All values in this paper are given with their s.E. of mean. Measurement of the potential difference (Barry, Dikstein, Matthews, Smyth & Wright, 1964) in Krebs-Henseleit (1932) bicarbonate-saline buffer containing 10 mm glucose mucosally gave a value of 5-32 + 0-46 (8) mV. In the same buffer, manometric determination of respiration (Umbreit, Burris & Stauffer, 1949) gave a QO, value of 3-7 ± 0 4 (5). Also in the same buffer,

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mucosal water transport defined as the amount in mg of water transferred from the mucosal solution was 90-0 + 16-0 (5) mg/200 mg tissue wet weight per hr and mucosal glucose transport defined as the amount in ug of glucose transferred from the mucosal solution was 99-5 ± 5.7 (10) jtg/mg tissue dry weight per hr. The final glucose concentration ratio when 10 mm glucose was included serosally was 1P66 + 0 34 (4). These values are comparable to those normally found in the proximal jejunum (Barry, Matthews & Smyth, 1961; Bronk & Parsons, 1965) and indicate that the sacs so prepared were viable and physiologically functional. (a) Measurement of pH in bicarbonate buffer. Acidification was measured in KrebsHenseleit (1932) buffer in a closed system because of the carbon dioxide component of the buffer. As it might affect the electrode response and alter pH readings, paraffin oil was not used in the procedure for measuring pH as other workers have done (Wilson & Kazyak, 1957). Instead, the pH was measured at a lower temperature at which the carbon dioxide is more soluble. Consequently, 10 ml. buffer in 25 ml. flasks were gassed with 95% 02:5% CO2 (V/V) (or 95 % N2:5 % CO2 (V/v) in anoxic experiments) whilst standing in iced water. Flasks were gassed for an hour to achieve full saturation. The fluid was then poured into a 20 ml. test-tube and the pH measured by a Pye 401 glass electrode coupled to a Pye-Dynacap 11087 pH meter. The fit between the walls of the test-tube and the glass electrode was such that almost no surface area for carbon dioxide loss presented itself. A drift to more alkaline pH values was detectable only after standing for 2-3 min and justified this method of measuring pH without transference under oil since measurement was completed within seconds. After measurement the fluid was returned to the flask. Sacs were made, placed in flasks and when the final sac had been made, after about 15 min, the flasks were stoppered, transferred to the water-bath and incubated. Adding the sacs to fluid at low temperature ruled out variations due to the time taken to prepare each individual sac since metabolism would be substantially reduced until the temperature was elevated. In this closed system, flasks were shaken (74 oscillations/min, amplitude 4 cm) for 1 hr in a shaking incubator at 370 C. After incubation, flasks were left to stand in iced water for a further hour to allow re-equilibration of carbon dioxide to that temperature, then opened and the pH measured again. In each experiment, flasks were run containing medium only. This acted as a measure of the error implicit in the procedure against which changes in pH due to carbon dioxide loss from the flask could be corrected. Where ATP was used and hydrolysis might have occurred owing to physical causes, control flasks, without tissue, containing 10 mM-ATP were compared with those without ATP and no difference in inherent carbon dioxide loss was found. (b) Measurement of pH in phosphate buffer. Acidification was also measured in an open system in phosphate buffer (Krebs & Henseleit, 1932) without the additional period on ice either before or after incubation to check for any untoward effects of chilling the tissue. The buffer was gassed with the same gas mixture as used in the bicarbonate buffer experiments but unlike the previous method, the gassing was continuous during the incubation at 370 C. Gassing was for 1 hr before experiment to achieve a stable pH. The pH was measured as previously described and the sacs made in the usual manner. With continuous gassing, additional shaking was superfluous since adequate mixing was ensured by the turbulence from the gas stream. After one hour's incubation at 370 C, the mucosal fluid was removed and the pH measured. Expression of result. At the end of the experiment, the serosal contents were drained and the ligatures dissected from the sacs which were dried to a constant weight at 80°C over a 48 hr period. The dry weight percentage of the total wet weight was approximately 15 %. In both phosphate and bicarbonate buffer experiments,

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actual hydrogen ion production per milligram tissue dry weight was calculated with the aid of a nomogram. Bicarbonate buffer was titrated against 0-1 N-HCl so that a change in buffer pH could be empirically related to the amount of titrated hydrogen ion needed to cause that change. Over the limited pH range 7-08-0 the pH response of the two buffers to increments of hydrogen ion was essentially the same. Acidification was expressed as jug hydrogen ion per mg tissue dry weight per hour and given as the mean + S.E. of the mean. Comparison between both methods. Often used in in vitro studies (Rasmussen, Waldorf, Dziewatkowski & DeLuca, 1963; Bronk & Parsons, 1965; Bronk & Leese, 1973; Blair et al. 1974), brief cooling of tissue might lead to leakage of potassium ion due to cessation of normal pumping activities, as is seen in red blood corpuscles (Maizels, 1949). Subsequent acidification at 370 C might in part derive from ion movements restoring the status quo. However, potassium ion loss in the presence of various hexoses in no way correlates with the presented data on acidification in the presence of similar hexoses (Brown & Parsons, 1962) ruling out a compulsory hydrogen: potassium ion exchange in this tissue. Furthermore, even at 00 C potassium ion pumping into cells is still maintained (Evans, Wrigglesworth, Burdett & Pover, 1971). Since acidification rates are the same in both buffers (Table 1), one of which involved no cooling procedure at all, there is little compelling evidence to suggest that low temperatures either prior to or after incubation induce acidification. The bicarbonate buffer procedure was preferred because although acidification was the same in both buffers, glucose transfer was greater (Table 1) and least loss of structural and functional integrity occurs in bicarbonate buffer (Benet et al. 1971; Parsons, 1971). Isotopic experiments. Two sacs were taken from each rat, after ether anaesthesia, and incubated in Krebs-Henseleit bicarbonate buffer for 30 min without agitation but with continuous gassing with 95: 5 % 02 :C02 (v/v). Both sets of sacs were incubated in this medium which also contained 10 mM-ATP with ['4C]ATP isotope, uniformly labelled in the sugar ring, of specific activity 10 #,c/m-mole; one set of sacs was incubated at 0° C, the other at 370 C. Mucosal aliquots (0-2 ml.) were taken prior to the experiment. After the incubation period, each sac was washed sequentially with three changes of 50 ml. of 154 mm saline. 0 2 ml. serosalfluid was then taken for counting. Each sac was then homogenized in 1 ml. of saline and a homogenate aliquot (0-2 ml.) was taken after deproteination by heating for 10 min at 90° C with subsequent centrifugation at 3000 rev/min. The aliquots were added to 10 ml. NE220 liquid scintillator. All such radioactive samples were counted on a Nuclear Enterprises 8305 scintillation spectrometer operating at 00 C. Counting efficiency was determined by internal standardization using n-1-'4C hexadecane 1-10 1ac/g as a standard. The tissue concentration of isotopic ATP was calculated on the basis of tissue water assuming that this amounted to 85 0/0 of the tissue wet weight. Chemical estimations. Lactic acid content of the mucosal and serosal fluids was assayed by the method of Hohorst (1957). Glucose was measured using the automated 'Technicon' autoanalyser procedure (Salway, 1969). RESULTS

Mucosal glucose (Table 2) caused an increase in acidification in the proxitual jejunum at concentrations of 1-0 mm (P < 0.05) and above (P < 0.01) to reach a limiting value between 1 and 10 mm. Although contrary to previous reports (Barry, Jackson & Smyth, 1966), acidification also occurred ill the absence of glucose. The changes in pH observed were not simply

ACIDIFICATION IN RAT PROXIMAL JEJUNUM 337 related to increased water transport due to addition of glucose. Mucosal water transfer from 10 ml. solution (Table 2) was measured during these experiments and found to be able to cause maximal errors of only 2 % in the estimation of hydrogen ion concentration. The essentially hyperbolic relation of acidification to increasing mucosal glucose concentration was demonstrated by a log transformation which gave a highly significant (P < 0 001) correlation (r = 0.997). A less significant (P < 0 05, r = 0-810) Woolf plot transformation (a better transformation of Michaelis-Menten 08

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Acidification in the rat proximal jejunum.

J. Physiol. (1975), 245, pp. 333-350 With 3 text-ftgure8 Printed in Great Britain 333 ACIDIFICATION IN THE RAT PROXIMAL JEJUNUM BY J. A. BLAIR, M...
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