Effect of E. coli heat-stable enterotoxin on colonic transport in guanylyl cyclase C receptor-deficient mice.

We studied the functional importance of the colonic guanylyl cyclase C (GCC) receptor in GCC receptor-deficient mice. Mice were anesthetized with pentobarbital sodium, and colon segments were studied in Ussing chambers in HCO3- Ringer under short-circuit conditions. Receptor-deficient mouse proximal colon exhibited similar net Na+ absorption, lower net Cl- absorption, and a negative residual ion flux (J(R)), indicating net HCO3- absorption compared with that in normal mice. In normal mouse proximal colon, mucosal addition of 50 nM Escherichia coli heat-stable enterotoxin (STa) increased the serosal-to-mucosal flux of Cl- (J(s-->m)(Cl)) and decreased net Cl- flux (J(net)(Cl)) accompanied by increases in short-circuit current (I(sc)), potential difference (PD), and tissue conductance (G). Serosal STa had no effect. In distal colon neither mucosal nor serosal STa affected ion transport. In receptor-deficient mice, neither mucosal nor serosal 500 nM STa affected electrolyte transport in proximal or distal colon. In these mice, 1 mM 8-bromo-cGMP produced changes in proximal colon J(s-->m)(Cl) and J(net)(Cl), I(sc), PD, G, and J(R) similar to mucosal STa addition in normal mice. We conclude that the GCC receptor is necessary in the mouse proximal colon for a secretory response to mucosal STa.

Effects of short chain fatty acids on colonic Na+ absorption and enzyme activity.

Short chain fatty acids (SCFA) stimulate colonic Na+ absorption and inhibit cAMP and cGMP-mediated Cl- secretion. It is uncertain whether SCFA have equivalent effects on absorption and whether SCFA inhibition of Cl- secretion involves effects on mucosal enzymes. Unidirectional Na+ fluxes were measured across stripped colonic segments in the Ussing chamber. Enzyme activity was measured in cell fractions of scraped colonic mucosa. Mucosal 50 mM acetate, propionate, butyrate and poorly metabolized isobutyrate stimulated proximal colon Na+ absorption equally (300%). Neither 2-bromo-octanoate, an inhibitor of beta-oxidation, nor carbonic anhydrase inhibition affected this stimulation. All SCFA except acetate stimulated distal colon Na+ absorption 200%. Only one SCFA affected proximal colon cGMP phosphodiesterase (PDE) (18% inhibition by 50 mM butyrate). All SCFA at 50 mM stimulated distal colon cAMP PDE (24-43%) and decreased forskolin-stimulated mucosal cAMP content. None of the SCFA affected forskolin-stimulated adenylyl cyclase in distal colon or ST(a)-stimulated guanylyl cyclase in proximal colon. Na+-K+-ATPase in distal colon was inhibited 23-51% by the SCFA at 50 mM. We conclude that all SCFA (except acetate in distal colon) stimulate colonic Na+ absorption equally, and the mechanism does not involve mucosal SCFA metabolism or carbonic anhydrase. SCFA inhibition of cAMP-mediated secretion may involve SCFA stimulation of PDE and inhibition of Na+-K+-ATPase.

Effect of short-chain fatty acids on cyclic 3',5'-guanosine monophosphate-mediated colonic secretion.

Short chain fatty acids (SCFA) prevent and reverse cyclic 3',5'-adenosine monophosphate (cAMP) but not Ca(2+)-mediated Cl- secretion. Mucosal [HCO3-]i has an opposite effect on these secretagogues. We examined whether SCFA and [HCO3-]i affect cyclic 3',5'-guanosine monophosphate (cGMP)-induced secretion. Stripped segments of male Sprague-Dawley rat (Rattus norvegicus) proximal and distal colon, and cultured T84 cells were studied in Using chambers, and pHi and [HCO3-]i were determined. Mucosal [cGMP] was measured in proximal colon. In T84 cells, the increase in Cl- secretion (measured as Isc) induced by mucosal 0.25 microM Escherichia coli heat-stable enterotoxin (STa) was prevented/reversed by bilateral 50 mM Na+ butyrate (71%/73%), acetate (58%/76%), propionate (68%/73%) and (poorly metabolized) isobutyrate (80%/79%). In proximal colon in HCO3- Ringer, basal Cl- secretion was not affected by [HCO3-]i or 25 mM butyrate. Mucosal 0.25 microM STa decreased net Na+ and Cl- absorption. Bilateral but not mucosal 25 mM SCFA reversed STa-induced effects on Na+ absorption and Cl- secretion. Bilateral and mucosal 25 mM SCFA but not [HCO3-]i prevented STa-induced Cl- secretion and increases in mucosal [cGMP]. STa did not produce Cl- secretion in distal colon. It was concluded that SCFA but not [HCO3-]i can prevent and reverse cGMP-induced colonic Cl- secretion.

Nonionic diffusion of short-chain fatty acids across rat colon.

Short-chain fatty acid (SCFA) transport across the colon may occur by nonionic diffusion and/or via apical membrane SCFA-/HCO3- exchange. To examine the relative importance of these processes, stripped segments of rat (Ratus ratus) proximal and distal colon were studied in Ussing chambers, and the unidirectional fluxes of radiolabeled SCFA butyrate, propionate, or weakly metabolized isobutyrate were measured. In N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) or 1 or 5 mM HCO3- Ringer, decreases in mucosal pH stimulated mucosal-to-serosal flux (Jm-->s) of all SCFA, decreases in serosal pH stimulated serosal-to-mucosal flux (Js-->m), and bilateral pH decreases stimulated both fluxes equally. These effects were observed whether the SCFA was present on one or both sides of the tissue, in both proximal and distal colon, in the absence of luminal Na+, and in the presence of either luminal or serosal ouabain. Changes in intracellular pH or intracellular [HCO3-] did not account for the effects of extracellular pH. Luminal Cl- removal, to evaluate the role of apical membrane Cl-/SCFA- exchange, had no effect on Jm-->s but decreased Js-->m 32% at pH 6.5 and 22% at 7.2. Increasing SCFA concentration from 1 to 100 mM, at pH 6.4 or 7.4, caused a linear increase in Jm-->s. We conclude that SCFA are mainly transported across the rat colon by nonionic diffusion.

Modulation of chloride secretion in the rat ileum by intracellular bicarbonate.

Increasing intracellular bicarbonate concentration ([HCO3-]i) inhibits calcium-mediated Cl- secretion in rat distal colon and T84 cells. We investigated the effect of [HCO3-]i on Cl- secretion in rat ileum. Segments of intact ileum from Sprague-Dawley rats were studied in Ussing chambers and villus and crypt intracellular pH and [HCO3-]i were determined using BCECF. A range of crypt and villus [HCO3-]i from 0 to 31 mM was obtained by varying Ringer's composition. Basal serosal-to-mucosal Cl- flux (JsmCl) averaged 8.5 +/- 0.2 mu eq.h-1.cm-2 and was unaffected by changing [HCO3-]i or serosal bumetanide. Carbachol increased JsmCl by 3.9 +/- 0.5 mu eq.h-1.cm-2 at [HCO3-]i = 0 mM but only by 1.0 +/- 0.3 mu eq.h-1.cm-2 at high crypt and villus [HCO3-]i. Dibutyryl-cAMP increased JsmCl by 2.5 +/- 0.2 mu eq.h-1.cm-2 at all [HCO3-]i. Carbachol and db-cAMP showed mutual antagonism at low [HCO3-]i and near-additivity at high [HCO3-]i. We conclude that like rat colon and T84 cells, calcium-mediated but not cAMP-mediated Cl- secretion in the ileum is inhibited by increasing [HCO3-]i. Mutual antagonism between carbachol and db-cAMP at low [HCO3-]i was present in ileum and distal colon but not in T84 cells.

Short-chain fatty acids inhibit cAMP-mediated chloride secretion in rat colon.

Butyrate stimulates salt absorption in mammalian colon. We examined whether butyrate also affects Cl- secretion. Mucosal segments of distal colon of male Sprague-Dawley rats and T84 cells were studied in Ussing chambers. In control colon, 1 mM dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) increased short-circuit current (Isc) and serosal-to-mucosal Cl- flux (JsmCl) by 3.2 +/- 0.8 and 2.9 +/- 0.8 mueq.cm-2.h-1, respectively. Mucosal or serosal 25 mM butyrate prevented DBcAMP-induced increases in Isc and JsmCl. Four and eight millimolar butyrate caused half-maximal inhibition of the increases in JsmCl and Isc, respectively. Butyrate also inhibited basal JsmCl (by 2.0 +/- 0.4 mueq.cm-2.h-1) but not carbachol-mediated Cl- secretion. The relative inhibitory potency at 25 mM of other short-chain fatty acids (SCFA) paralleled their degree of cellular metabolism: butyrate > acetate = propionate > isobutyrate. At 25 mM, all SCFA reduced mucosal intracellular pH (pHi) transiently by 0.1 pH unit. In intact T84 cells, 50 mM butyrate inhibited the DBcAMP-induced rise in Isc by 55%. In T84 cells with nystatin-permeabilized basolateral membranes, butyrate inhibited the increase in Isc by 82%. We conclude that butyrate inhibits basal and cAMP-mediated Cl- secretion by a mechanism independent of pHi, possibly located at the apical membrane.

Dissociation of colonic apical Na/H exchange activity from bulk cytoplasmic pH.

Intracellular acidification by stimuli rather than CO2 fails to stimulate colonic apical Na/H ex-change and Na absorption. We examined whether Na absorption could be stimulated in the absence of changes in cytoplasmic pH (pHi). Distal colon of male Sprague-Dawley rats was used for pHi measurements with 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein and for flux measurements in Ussing chambers. In 21 mM HCO3-Ringer, increasing PCO2 from 20 to 70 mmHg decreased pHi from 7.51 to 7.03 and increased net Na flux (JnetNa) from 4.2 +/- 0.4 to 6.8 +/- 0.6 mu eq.cm-2.h-1. Similar increases in JnetNa occurred in the absence of mucosal CI and in the presence of phalloidin to inhibit microfilaments or penzolamide to inhibit membrane-bound carbonic anhydrase. sohydric increases in Pco2 did not alter pHi but stimulated JnetNa from 5.1 +/- 0.6 to 7.2 +/- 0.8 mu eq.cm-2.h-1. Carbonyl cyanide m-chlorophenylhydrazone (CCCP) decreased pHi from 7.45 to 7.35 but did not stimulate JnetNa. Butyrate (25 mM) decreased pHi from 7.15 to 7.02 with recovery to baseline within 6 min; however, JnetNa increased by 2.2 mu eq.cm-2.h-1 for 60 min. We conclude that apical Na/H exchange activity is unresponsive to changes in bulk pHi and is independent of Cl/HCO3 exchange, microfilaments, and membrane-bound carbonic anhydrase. The presence of an H-tight, CO2, and butyrate-permeable subapical domain is postulated.

Modulation of secretagogue-induced chloride secretion by intracellular bicarbonate.

We have previously demonstrated inhibition of basal Cl- secretion by intracellular bicarbonate concentration ([HCO3-]i) in rat distal colon. We now examined whether secretagogue-induced Cl- secretion is inhibited by [HCO3-]i as well. Stripped segments of distal colon from male Sprague-Dawley rats and the colon tumor cell line T84 were studied. Flux measurements were performed in the Ussing chamber under short-circuit conditions. [HCO3-]i was calculated from intracellular pH (pHi) values that were estimated with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP) and carbachol were used as secretagogues. In both distal colon and T84 cells, [HCO3-]i did not affect cAMP-induced Cl- secretion. However, carbachol-induced secretion was inhibited by [HCO3-]i; in rat colon, Cl- secretion decreased from 2.3 to 1.5 mueq.cm-2.h-1 when [HCO3-]i was increased from 15.0 to 28.4 mM (P < 0.05). In T84 cells, the change in short-circuit current decreased from 8.1 to 1.1 microA/cm2 over a range of [HCO3-]i from 0 to 15.6 mM (P < 0.001). We conclude that [HCO3-]i is an important modulator of carbachol-stimulated Cl- secretion in both rat distal colon and the T84 cell line. cAMP-mediated secretion is not affected by [HCO3-]i.

Effect of intracellular acidification on colonic NaCl absorption.

CO2 stimulates Na+ and Cl- absorption in rat distal colon. This is most likely due to intracellular generation of H+ and HCO3- and stimulation of apical Na(+)-H+ and Cl(-)-HCO3- exchangers. We examined whether intracellular acidification by means other than CO2 would also stimulate Na+ absorption. Stripped segments of distal colon from male Sprague-Dawley rats were studied under short-circuit conditions in Ussing chambers. Identically prepared tissues were used for intracellular pH (pHi) measurements with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. When the Ringer PCO2 was increased from 20 to 34 mmHg, pHi decreased from 7.50 +/- 0.04 to 7.35 +/- 0.04 and net Na absorption increased from 2.4 +/- 0.7 to 3.7 +/- 0.7 mu eq.cm-2 x h-1. A similar degree of intracellular acidification was obtained with 2.6 microM nigericin, but no stimulation of Na+ absorption was seen. When Ringer-HCO3- concentration was reduced from 39 to 11 mM at constant PCO2 = 35 mmHg, pHi decreased from 7.55 +/- 0.02 to 7.11 +/- 0.02 with no effect on net Na+ absorption. A similar reduction in pHi in a CO2-HCO3(-)-free, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered Ringer also did not stimulate Na+ absorption. Methazolamide had no effect on steady-state pHi at any given PCO2 but caused marked reductions in net Na+ absorption (9.6 +/- 2.4 to 5.2 +/- 1.2 mu eq.cm-2 x h-1 at PCO2 = 70 mmHg). We conclude that Na+ absorption in rat distal colon is not stimulated by intracellular acidification per se but rather has an absolute requirement for CO2 and carbonic anhydrase activity.

Modulation of chloride secretion in the rat colon by intracellular bicarbonate.

Extracellular HCO3- stimulates colonic net Cl- absorption in part by inhibiting basal Cl- secretion. This inhibition was investigated by measuring serosal-to-mucosal Cl- flux across short-circuited colonic segments from Sprague-Dawley rats. Mucosal intracellular pH and bicarbonate were estimated using the pH-sensitive dye BCECF. When extracellular [HCO3-] ([HCO3-]e) was increased from 0 to 39 mmol/L at PCO2 33 mm Hg, mucosal intracellular [HCO3-] ([HCO3-]i) increased to 25.3 mmol/L and serosal-to-mucosal Cl- flux decreased from 13.0 to 7.1 microEq.cm-2.h-1. When PCO2 was increased to 72 mm Hg at [HCO3-]e 39 mmol/L, [HCO3-]i increased to 29.8 mmol/L and serosal-to-mucosal Cl- flux decreased to 5.9 microEq.cm-2.h-1. In Ringer's solution containing 21 mmol/L HCO3- and 20 mmol/L Cl- (but not 100 mmol/L Cl-), increasing PCO2 from 21 to 70 mm Hg increased [HCO3-]i to 22.6 mmol/L and decreased serosal-to-mucosal Cl- flux from 3.0 to 1.7 microEq.cm-2.h-1. Overall, serosal-to-mucosal Cl- flux was inversely related to [HCO3-]i on either side of an [HCO3-]i plateau of 9-18 mmol/L at which flux was stable. These data suggest that [HCO3-]i is an important modulator of basal Cl- secretion in rat distal colon.

Effect of CO2 on rat colonic Na absorption: studies with nystatin.

An increase in ambient CO2 tension from 3% to 11% augments colonic Na absorption in the rat. The membrane site of action of CO2 was examined by measuring colonic Na absorption in the Ussing chamber when nystatin was used to permeabilize the luminal (apical) membrane. The equal rates of ouabain-sensitive Na absorption at 3% and 11% CO2 in the presence of nystatin and at 11% CO2 in its absence suggested that CO2 acted at the luminal membrane. This finding was also observed at a submaximal rate of Na absorption (produced by lowering bathing solution Na from 140 to 27 mmol/L) and in a Cl-free solution (to prevent cell swelling). The basolateral membrane was indeed rate limiting for Na absorption in the presence of nystatin, because methylprednisolone (3 mg/kg SC for 3 days to increase sodium-potassium--stimulated adenosine triphosphatase activity) increased Na absorption measured in the presence of nystatin and because CO2 increased absorption in steroid-treated rats in the absence of nystatin. These results validate the protocol and confirm the luminal site of action of CO2 and nystatin on colonic Na absorption.

NaCl absorption in the rabbit ileum. Effect of acid-base variables.

In vivo and in vitro studies suggest that acid-base variables regulate ion transport in the rabbit ileum. The relative importance of these variables on active Na+ and Cl- absorption has not been defined. Isolated, stripped ileal segments were studied under short-circuited conditions in the Ussing flux chamber. Unidirectional 22Na and 36Cl fluxes were measured after changes in bathing solution pH, PCO2, and/or [HCO3-]. When pH was decreased from 7.6 to 7.1, net flux of Na+ increased from 0.1 +/- 0.7 to 2.6 +/- 0.7 mu Eq/cm2 per hour and net flux of Cl- increased from -2.0 +/- 0.9 to 1.3 +/- 0.6 mu Eq/cm2 per hour. These changes were rapid in onset, completely reversible, and accounted for by changes in mucosal-to-serosal fluxes of these ions. They were accompanied by small decreases in short-circuit current, but there were no changes in residual flux. These pH effects were not inhibited by the presence of luminal bumetanide (1 mmol/L), furosemide (1 mmol/L), hydroflumethiazide (1 mmol/L), or 4,4'-diisothiocyanostilbene-2,2'-disulfonate (1 mmol/L), or by the carbonic anhydrase inhibitor methazolamide (1 mmol/L). When data from all combinations of acid-base conditions were combined and analyzed by linear regression, pH was the only variable that correlated with mucosal-to-serosal fluxes (r = -0.84) and net flux (r = -0.85) for Na+, mucosal-to-serosal fluxes (r = -0.96) and net flux (r = -0.99) for Cl-, and short-circuit current (r = 0.97). These findings suggest that extracellular pH modulates active Na+ and Cl- absorption in the rabbit ileum.

Effects of pH and cyclic adenosine monophosphate on ileal electrolyte transport in the rat and rabbit.

Alterations in extracellular pH cause reciprocal changes in NaCl absorption in the rat and rabbit ileum. The presence of cholera toxin-induced secretion does not affect pH action measured by in vivo perfusion of the rat ileum. We examined the interaction of pH and cyclic adenosine monophosphate-induced secretion in the rabbit ileum. We found that alterations in arterial pH did not affect ileal absorption in the rabbit in the presence of cholera toxin-induced secretion. This was true whether transport was studied during in vivo ileal perfusion of anesthetized rabbits or by measuring Na+ and Cl- fluxes across isolated, short-circuited tissues in the Ussing chamber. The effects of pH also were blocked when normal rabbit ileum was exposed to 1 mmol/L dibutyryl cyclic adenosine monophosphate in vitro. By contrast, alterations in bathing solution pH affected ileal absorption in the rat in the presence and absence of cyclic adenosine monophosphate. Similarly, exposure to cyclic adenosine monophosphate did not affect the response of the rat colon to PCO2. These findings suggest that the apparently independent effects of pH and cyclic adenosine monophosphate in the rat ileum are not universal. In tissues such as the rabbit ileum, the mechanisms of pH and cyclic adenosine monophosphate action may have biochemical or physiological pathways in common.

Membrane site of action of CO2 on colonic sodium absorption.

Increases in ambient CO2 tension increase colonic sodium absorption by increasing mucosal to serosal sodium flux. We examined the membrane site of CO2 action by utilizing the polyene antibiotic nystatin to create aqueous pores in the apical membrane. Under these conditions, the basolateral rather than the apical membrane is rate limiting for sodium absorption. Pairs of stripped rat distal colonic segments were mounted in modified Ussing chambers in a Ringer-HCO3 solution gassed with either 3% CO2-97% O2 or 11% CO2-89% O2. Mucosal-to-serosal 22Na and 36Cl fluxes were measured under short-circuited conditions, and ouabain-sensitive absorption was calculated before and after the addition of mucosal nystatin 300 U/ml. Ouabain-sensitive sodium absorption was fivefold greater at 11% CO2 than at 3% CO2 before nystatin addition. Nystatin increased short-circuit current (Isc), transcolonic conductance (Gt) and ouabain-sensitive sodium absorption at 3% CO2 but only increased Isc and Gt at 11% CO2. The levels of sodium absorption at 3% and 11% CO2 after nystatin were equal and identical to the level measured at 11% CO2 in the absence of nystatin. Ouabain-sensitive chloride absorption was similar at 3% and 11% CO2 in the absence of nystatin and was not affected by nystatin addition. These findings suggest that ambient CO2 tension affects colonic sodium absorption by a selective action at the apical membrane.

Methodological errors in radioisotope flux measurements.

We examined several sources of error in isotopic flux measurements in a commonly used experimental model: the study of 22Na and 36Cl fluxes across rat ileal tissue mounted in the Ussing flux chamber. The experiment revealed three important sources of error: the absolute counts per minute, the difference in counts per minute between serial samples, and averaging of serial samples. By computer manipulation, we then applied hypothetical changes in the experimental protocol to generalize these findings and assess the effect and interaction of the absolute counts per minute, the sampling interval, and the counting time on the magnitude of the error. We found that the error of a flux measurement will vary inversely with the counting time and the difference between the consecutive sample counts per minute used in the flux calculations and will vary directly with the absolute counts per minute of each sample. Alteration of the "hot" side specific activity, the surface area of the tissue across which flux is measured and the sample volume have a smaller impact on measurement error. Experimental protocols should be designed with these methodological considerations in mind to minimize the error inherent in measuring isotope flux.

Effects of acid-base variables on ion transport in rat colon.

Alterations in arterial acid-base variables have important effects on colonic electrolyte transport in vivo. To confirm the relative effects of these variables and to characterize the transport processes involved, we measured unidirectional 22Na and 36Cl fluxes across short-circuited, distal colonic mucosa of Sprague-Dawley rats. Stripped tissues were studied in Hepes buffer and in Ringer's solutions at HCO3 concentrations of 11, 21, and 39 mM, and CO2 tensions between 0 and 69.6 mmHg. Increases in PCO2, but not in either pH or HCO3 concentration, caused similar increases in JNanet and JClnet (net flux of sodium and chloride, respectively) from -0.2 +/- 0.3 and -1.5 +/- 0.4 mu eq/cm2 per h at PCO2 = 0 to 6.8 +/- 0.6 and 7.6 +/- 0.7 mu eq/cm2 per h, respectively, at PCO2 = 69.6 mmHg. These increases were accounted for by changes in Jms and were accompanied by small decreases in Isc. 1 mM acetazolamide decreased both JNanet and JClnet and their responses to increases in CO2. 0.75 mM luminal amiloride prevented the increase in sodium absorption, but did not affect the CO2-induced increase in chloride absorption. In the presence of amiloride, CO2 increased JR (residual flux). 0.1 mM luminal furosemide did not affect the CO2-induced increases in JNanet in the absence or presence of amiloride. Changes in HCO3 concentration did not alter JR. We conclude that ambient CO2 effects active, electroneutral sodium absorption in the rat distal colon. The process stimulated by CO2 is dependent on mucosal carbonic anhydrase activity and most likely represents Na/H and Cl/HCO3 ion exchange.

Changes in cAMP phosphodiesterase activity during oral mucosal regeneration.

Preliminary studies (Lineweaver-Burk, Ca2+ calmodulin sensitivity) suggest that oral mucosa contains at least two cyclic AMP phosphodiesterases, one a high affinity (low Km) enzyme and the other a low affinity (high Km) enzyme. Analysis of the distribution of both enzymatic forms during oral mucosal regeneration revealed that the low Km, and high Km cAMP phosphodiesterase activities were significantly elevated prior to the first wave of cAMP accumulation and during the second cAMP wave. Although the cAMP peaks declined between 20-24 h, both cAMP phosphodiesterases remained significantly elevated at the wound site. The apparent Km of the low Km form increased from 5.3 to 7.5 microM, while that of the high Km form remained essentially unaltered 20 h after wounding. The low Km, and high Km cAMP phosphodiesterase activities in normal rat oral mucosa were not affected by epinephrine or insulin; were slightly inhibited by glucagon, and significantly inhibited by methylprednisolone. Imidazole and histamine activated both forms and theophylline was inhibitory to the enzyme. The high Km cAMP phosphodiesterase was sensitive, and the low Km form insensitive to Ca2+ calmodulin stimulation.

Effect of inflammation upon human gingival cyclic AMP metabolism.

The effect of the increasing degree of human gingival inflammation on adenylate cyclase (basal, fluoride stimulated) and low Km and high Km cAMP phosphodiesterase activities were evaluated in separate studies. Human gingival biopsies were classified by the Löe Bleeding Index as mildly, moderately, and markedly inflamed. Basal and F- stimulated adenylate cyclase (cAMP synthesis) activities were found to be unaltered by the increasing degree of inflammation when the data were expressed on either a mg wet wt, or mg protein basis. A significant loss of F- stimulated adenylate cyclase (mg protein) activity was observed in the moderately inflamed group when the data were compared with either the mildly or markedly inflamed groups of tissue. The low Km, and high Km cAMP phosphodiesterase activities (cAMP degradation) were found to be unaffected by gingival inflammation. This suggests that neither cAMP synthesis, nor degradation are stimulated in human gingiva by inflammation.

Adenylate cyclase localization in unfixed specimens of rat oral mucosa and isolated mitochondria.

The cytochemical localization of adenylate cyclase (E.C.4.6.1.1) was demonstrated in unfixed rat oral mucosal specimens and isolated mitochondrial fractions by use of a modified cytochemical reaction mixture. The adenylate cyclase reaction product was observed in the mitochondrial cristae of intact epithelial and fibroblast cells, and in isolated mitochondria after a 10-min incubation in the reaction mixture. The validity of the cytochemical adenylate cyclase method was supported by biochemical studies.

Elastase +/- soybean trypsin inhibitor dissociation of rat oral mucosa: ultrastructural and oxidative metabolic destructive changes in isolated, epithelial and dermal mitochondria after dissociation.

Epithelial and connective tissue compartments of rat oral mucosa were dissociated after incubation with elastase +/- soybean trypsin inhibitor (SBTI). Elastase + SBTI induced greater ultrastructural damage within the dissociated compartments than elastase alone. The basal lamina remained with the epithelial layer after elastase separation and was destroyed after exposure to elastase + SBTI. Isolated epithelial mitochondria were more severely damaged ultrastructurally after elastase + SBTI separation of the compartment than those prepared after exposure to elastase alone. Isolated fibroblast mitochondria were damage to the same extent after dissociation of the compartment with either medium. Oxidative metabolism and mitochondrial recoveries declined significantly after exposure to either dissociating medium. Cytochrome oxidase activity was significantly greater than succinic cytochrome c reductase in the control and experimental groups. Oxidative metabolism was found to be significantly greater in the connective tissue compartment than the epithelial compartment after dissociation of immature rat oral mucosa. Our data suggests that caution be utilized in assessing cellular viability and oxidative metabolism in tissue compartments immediately after their dissociation by proteolytic enzymes.