Stimulation of Cl(-) secretion by chlorzoxazone

We previously demonstrated that 1-ethyl-2-benzimidazolone (1-EBIO) directly activates basolateral membrane calcium-activated K(+) channels (K(Ca)), thereby stimulating Cl(-) secretion across several epithelia. In our pursuit to identify potent modulators of Cl(-) secretion that may be useful to overcome the Cl(-) secretory defect in cystic fibrosis (CF), we have identified chlorzoxazone [5-chloro-2(3H)-benzoxazolone], a clinically used centrally acting muscle relaxant, as a stimulator of Cl(-) secretion in several epithelial cell types, including T84, Calu-3, and human bronchial epithelium. The Cl(-) secretory response induced by chlorzoxazone was blocked by charybdotoxin (CTX), a known blocker of K(Ca). In nystatin-permeabilized monolayers, chlorzoxazone stimulated a basolateral membrane I(K), which was inhibited by CTX and also stimulated an apical I(Cl) that was inhibited by glibenclamide, indicating that the G(Cl) responsible for this I(Cl) may be cystic fibrosis transmembrane conductance regulator (CFTR). In membrane vesicles prepared from T84 cells, chlorzoxazone stimulated (86)Rb(+) uptake in a CTX-sensitive manner. In excised, inside-out patches, chlorzoxazone activated an inwardly-rectifying K(+) channel, which was inhibited by CTX. 6-Hydroxychlorzoxazone, the major metabolite of chlorzoxazone, did not activate K(Ca), whereas zoxazolamine (2-amino-5-chlorzoxazole) showed a similar response profile as chlorzoxazone. In normal human nasal epithelium, chlorzoxazone elicited hyperpolarization of the potential difference that was similar in magnitude to isoproterenol. However, in the nasal epithelium of CF patients with the DeltaF508 mutation of CFTR, there was no detectable Cl(-) secretory response to chlorzoxazone. These studies demonstrate that chlorzoxazone stimulates transepithelial Cl(-) secretion in normal airway epithelium in vitro and in vivo, and suggest that stimulation requires functional CFTR in the epithelia.     

Inhibition of thromboxane A(2)-induced Cl(-) secretion by antidiarrhea drug loperamide in isolated rat colon

The antitumor drug irinotecan clinically causes severe diarrhea as a side effect. Thromboxane A(2) (TXA(2)), released by irinotecan, has been shown to be a novel physiological stimulant of Cl(-) secretion in the rat colon. Herein, we examined the effect of loperamide, an antidiarrhea drug, on Cl(-) secretion induced by irinotecan; 9, 11-epithio-11,12-methano-thromboxane A(2) (STA(2)), a stable TXA(2) analog; and prostaglandin E(2) (PGE(2)) by using isolated mucosae of the rat colon. In the presence of atropine, loperamide in a concentration-dependent manner inhibited the Cl(-) secretion induced by irinotecan, STA(2), and PGE(2). However, the drug inhibited more effectively the irinotecan- and STA(2)-induced secretion (IC(50) = 0. 7 and 1.2 microM, respectively) than the PGE(2)-induced secretion (IC(50) = 23 microM). Naloxone, an opiate antagonist, did not affect the antisecretory action of loperamide. Similar to the case for loperamide, W-7, a specific calmodulin antagonist, inhibited more effectively the STA(2)-induced Cl(-) secretion (IC(50) = 5 microM) than the PGE(2)-induced secretion (IC(50) = 36 microM). W-5, a low-affinity calmodulin antagonist (a dechlorinated control analog of W-7), also inhibited the STA(2)-induced secretion, but this effect was much less than that of W-7. STA(2)-induced increase in the intracellular free Ca(2+) concentration of single colonic crypt cells was not affected by loperamide. We suggest that loperamide efficiently inhibits the TXA(2)-induced secretion by blocking the calmodulin system in the colonic epithelium. The present results may explain why coadministration of loperamide with irinotecan is clinically efficient for avoiding the irinotecan-induced side effect of diarrhea.     

Ca-mediated stimulation of Cl secretion by reactive oxygen metabolites in human colonic T84 cells.

Monochloramine (NH2Cl), a granulocyte-derived reactive oxygen metabolite (ROM), increases short-circuit current (Isc) in cultured T84 monolayers in a concentration-dependent manner up to nonlethal concentrations of 75 microM. Isc increases slowly after NH2Cl, reaching a peak value of 18 +/- 2 microA/cm2 20 min after addition. The Isc changes are persistent (lasting over 20-30 min), depend on medium Cl, and are inhibitable with bumetanide. 36Cl flux studies demonstrated that NH2Cl increases serosa-to-mucosa flux of Cl without changing mucosa-to-serosa flux, consistent with stimulation of electrogenic Cl secretion. Isc responses to NH2Cl, but not PGE2, are dependent on medium calcium. As demonstrated in fura-2-loaded T84 cells, NH2Cl increases free cytosolic calcium by influx of extracellular Ca2+ and by release of Ca2+ from endogenous stores. However, NH2Cl had no effect on phosphatidylinositol metabolism or cyclic nucleotide levels. We conclude that ROM directly stimulate electrolyte secretion, an effect in part mediated by increases in cytosolic Ca2+, possibly through increasing Ca2+ permeability of cellular membranes.     

Nucleotide-induced restoration of conjunctival chloride and fluid secretion in adenovirus type 5-infected pigmented rabbit eyes

We evaluated the role of extracellular UTP and other nucleotides in the regulation of chloride (JCl) and fluid secretion (JCl) across the pigmented rabbit conjunctiva. Jv was determined in freshly excised conjunctival tissues mounted between two buffer reservoirs maintained in an enclosed environment at 37 degrees C. Short circuit current (Isc) and 36Cl flux were measured using modified Ussing-type chambers. Fluid flux measurements were made with a pair of capacitance probes. After observing the baseline for 15 to 30 min, fluid flux was measured in the presence of mucosally applied nucleotides (10 microM) for a period of 30 min. Mucosal application of 10 microM each of UTP, UDP, ATP, ADP, AMP, adenosine, and ATP-gamma-S transiently stimulated fluid secretion across the conjunctiva to a significant extent for 10 to 15 min. Other nucleotides did not show any significant effect. The stimulation of fluid secretion correlated well with the stimulation in Isc (r2 = 0.85). UTP (0.1-1000 microM) led to a maximal increase in fluid secretion by 11.72 +/- 0.48 microl/(h x cm2) with an EC50 value of 10.39 +/- 1.08 microM. ATP (0.1-1000 microM) caused a maximal increase in fluid secretion by 11.89 +/- 0.88 microl/(h x cm2) with an EC50 value of 17.23 +/- 2.63 microM. Adenovirus type 5 (Ad5) infection significantly decreased both net 36Cl secretion across the conjunctiva by approximately 56% and the rate of fluid secretion by approximately 56%. UTP (10 microM), but not 1 mM 8-bromo-cAMP, was able to elicit a normal stimulatory response in the Ad5-infected tissues. In conclusion, mucosal application of purinergic nucleotides may be therapeutically important in restoring ion and fluid secretion in the diseased conjunctiva.     

Bisphenol A inhibits Cl(-) secretion by inhibition of basolateral K+ conductance in human airway epithelial cells

There has been growing concern about the potential threat of hormone-disrupting chemicals like bisphenol A to various aspects of animal and human health. We studied the effects of bisphenol A on the Cl(-) secretion in human airway epithelial Calu-3 cells. Pretreatment with bisphenol A (IC(50) = 60 microM, for 30 min) prevented isoproterenol (10 nM)-generated short-circuit current (I(sc)) more potently than 17beta-estradiol or tamoxifen (IC(50) = 1 mM). 5'-Nitro-2-(3-phenylpropylamino) benzoate-sensitive apical conductance potentiated by isoproterenol was not affected by the pretreatment with either of these estrogenic compounds. The effects of bisphenol A were simulated in I(sc) responses to forskolin (10 microM) and 8-bromo-cAMP (1 mM). Nystatin permeabilization of Calu-3 monolayers revealed that bisphenol A attenuated 8-bromo-cAMP-induced basolateral K+ current, which is sensitive to clotrimazole (30 microM) and insensitive to charybdotoxin (100 nM), without affecting the apical Cl(-) current. Bisphenol A, but neither 17beta-estradiol nor tamoxifen, interrupted the charybdotoxin-sensitive component of I(sc) stimulated by 1-ethyl-2-benzimidazolinone (1-EBIO; 500 microM). The inhibitory effects of bisphenol A on these Cl(-) secretory stimuli were remarkable when applied to the apical rather than the basolateral membrane. Alternatively, long-term incubation of bisphenol A (1 microM; 12-72 h) had no discernible effect on isoproterenol- and 1-EBIO-induced Cl(-) secretion. These findings indicate that short-term exposure to bisphenol A attenuates transepithelial Cl(-) secretion through inhibition of both cAMP- and Ca(2+)-activated K+ channels on the basolateral membrane, interacting from the cytosolic surface in Calu-3 cells.     

Antiflammins suppress the A23187- and arachidonic acid-dependent chloride secretion in rabbit distal colonic mucosa.

Antiflammins are synthetic peptides with sequence homology to proteins inhibitory for phospholipase A2. The effects of antiflammins on chloride secretion induced by the calcium ionophore A23187, arachidonic acid (AA) and prostaglandin E2 (PGE2) were investigated in distal rabbit colonic mucosa mounted in Ussing-type chambers. 100 nM AF-2 (antiflammin 2, peptide HDMNKVLDL) fully prevented the increase in Isc, net chloride secretion, tissue PGE2 and second messenger cAMP induced by 5 microM A23187. AF-1 (antiflammin 1, peptide MQMKKVLDS) also inhibited, although to a lesser extent, the A23187-mediated increase in Isc. AF-2 inhibition began at doses of 100 nM (delta Isc -0.20 +/- 0.08, microEq h-1 cm-2, mean +/- S.E.M., N = 3) and was maximal at doses comprised between 200 and 250 nM (delta Isc -0.51 +/- 0.12, microEq h-1 cm-2, mean +/- S.E.M., N = 3). AF-2 also inhibited the increase in Isc and chloride secretion induced by the incubation with 10 microM AA and bradykinin but it was ineffective when tissues were stimulated with 10 microM PGE2. 100 nM AF-2 brought about an inhibition of the increase in AA-mediated increases in PGs and cAMP comparable to that of 10 microM of the cyclooxygenase inhibitor indomethacin. In vitro assay of colonic cyclooxygenase activity showed that 100 nM AF-2 and AF-1 inhibited cyclooxygenase activity (73 and approximately 30%, respectively), but they did not modify the in vitro activity of porcine phospholipase A2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolic acidosis stimulates H+ secretion in the rabbit outer medullary collecting duct (inner stripe) of the kidney.

The outer medullary collecting duct (OMCD) absorbs HCO3- at high rates, but it is not clear if it responds to metabolic acidosis to increase H+ secretion. We measured net HCO3- transport in isolated perfused OMCDs taken from deep in the inner stripes of kidneys from control and acidotic (NH4Cl-fed for 3 d) rabbits. We used specific inhibitors to characterize the mechanisms of HCO3- transport: 10 microM Sch 28080 or luminal K+ removal to inhibit P-type H+,K+-ATPase activity, and 5-10 nM bafilomycin A1 or 1-10 nM concanamycin A to inhibit H+-ATPase activity. The results were comparable using either of each pair of inhibitors, and allowed us to show in control rabbits that 65% of net HCO3- absorption depended on H+-ATPase (H flux), and 35% depended on H+,K+-ATPase (H,K flux). Tubules from acidotic rabbits showed higher rates of HCO3- absorption (16.8+/-0.3 vs. 12.8+/-0.2 pmol/min per mm, P < 0.01). There was no difference in the H,K flux (5.9+/-0.2 vs. 5.8+/-0.2 pmol/min per mm), whereas there was a 61% higher H flux in segments from acidotic rabbits (11.3+/-0.2 vs. 7.0+/-0.2 pmol/min per mm, P < 0.01). Transport was then measured in other OMCDs before and after incubation for 1 h at pH 6.8, followed by 2 h at pH 7.4 (in vitro metabolic acidosis). Acid incubation in vitro stimulated HCO3- absorption (12.3+/-0.3 to 16.2+/-0.3 pmol/min per mm, P < 0.01), while incubation at pH 7.4 for 3 h did not change basal rate (11.8+/-0.4 to 11.7+/-0.4 pmol/min per mm). After acid incubation the H,K flux did not change, (4.7+/-0.4 to 4.6+/-0.4 pmol/min per mm), however, there was a 60% increase in H flux (6.6+/-0.3 to 10.8+/-0.3 pmol/min per mm, P < 0.01). In OMCDs from acidotic animals, and in OMCDs incubated in acid in vitro, there was a higher basal rate and a further increase in HCO3- absorption (16.7+/-0.4 to 21.3+/-0.3 pmol/min per mm, P < 0.01) because of increased H flux (11.5+/-0.3 to 15.7+/-0.2 pmol/min per mm, P < 0.01) without any change in H,K flux (5.4+/-0.3 to 5.6+/-0.3 pmol/min per mm). These data indicate that HCO3- absorption (H+ secretion) in OMCD is stimulated by metabolic acidosis in vivo and in vitro by an increase in H+-ATPase-sensitive HCO3- absorption. The mechanism of adaptation may involve increased synthesis and exocytosis to the apical membrane of proton pumps. This adaptation helps maintain homeostasis during metabolic acidosis.     

Central serotonergic stimulation of aldosterone secretion.

Serotonin stimulates aldosterone secretion both in vitro and in vivo, and serotonin antagonism decreases plasma aldosterone levels in patients with idiopathic aldosteronism. This study was designed to assess the effects of the serotonin precursor, 5-hydroxytryptophan (5HTP), upon aldosterone secretion in man, and to determine whether stimulatory effects of 5HTP are mediated through the central nervous system. Oral 5HTP, administered as a single 200-mg dose, increased plasma aldosterone levels from 4.7 +/- 0.6 to 13.3 +/- 2.8 ng/dl in dexamethasone-pretreated, normal volunteers. Peripheral inhibition of decarboxylation of 5HTP, achieved by pretreatment with carboxydopa, 25 mg three times daily for 3 d, significantly increased the stimulatory effects of 5HTP on aldosterone levels (P less than 0.001). No change in aldosterone levels occurred in subjects who received placebo after pretreatment with dexamethasone and carboxydopa. Increased aldosterone was not accompanied by increases in plasma levels of renin activity, potassium, or ACTH. Plasma levels of 5HTP were markedly increased by carboxydopa pretreatment, but peak plasma levels of serotonin were not significantly altered. Four patients with idiopathic aldosteronism all had an increase in plasma aldosterone levels after 5HTP administration, whereas the response in four patients with aldosterone-producing adenoma was variable. Incubation of isolated human and rat adrenal glomerulosa cells with serotonin resulted in increased aldosterone secretion by both sets of cells, whereas 5HTP was ineffective in stimulating aldosterone secretion in vitro. We conclude that central serotonergic pathways are involved in the stimulation of aldosterone induced by administration of 5HTP. This mechanism may be an important etiologic factor in the hypersecretion of aldosterone that occurs in patients with idiopathic aldosteronism.     

Inhibitory effect of ascorbic acid (vitamin C) on cortisol secretion following adrenal stimulation in children.

Measurements of plasma cortisol values before and at 4 and 6 hours after intramuscular administration of a depot preparation of synthetic b1-24 corticotrophin were carried out in 12 healthy children (group B) before, as well as on the 5th day of continuous ascorbic acid (AA) administration (1 g t.i.d. orally). Comparison of the results in group B with those of 8 healthy children similarly treated with corticotrophin but not given AA (group A) showed that, on the 5th day of AA administration the mean cortisol values after ACTH were significantly lower than the corresponding values in group A (p less than 0.02), or the post-ACTH values in group B observed on the 1st experimental day, i.e., before AA administration (p less than 0.001). On the other hand, AA administration had no significant effect on the fasting plasma cortisol values. These data suggest that AA excess following adrenal stimulation with ACTH exerts an inhibitory effect on cortisol secretion and consequently it may be of no benefit in conditions of stress.     

Mechanism of deoxycholic acid stimulation of the rabbit colon.

Previous studies showed that deoxycholic acid (DCA) stimulated migrating action potential complexes (MAPC) in the colon. The aim of this study was to clarify the mechanism of DCA-stimulated colonic motility. Myoelectrical and contractile activity were measured in New Zealand White rabbits from a loop constructed in the proximal colon. During the control period, slow waves were present at a frequency of 10.8 +/- 0.5 cycle/min and there were 1.5 +/- 0.5 MAPC/ h. After adding DCA (16 mM) to the loop the slow wave activity was unchanged. However, MAPC increased to 15.1 +/- 2.4 MAPC/h (P less than 0.001). MAPC activity was not stimulated in the colonic smooth muscle outside the loop. The intraluminal addition of procaine or tetrodotoxin to the colonic loop inhibited the DCA-stimulated increase in MAPC activity (0.2 +/- 0.2 MAPC/h) (P less than 0.005). Intravenous administration of atropine or phentolamine also inhibited MAPC activity that had been stimulated by DCA (P less than 0.005). Pretreatment with 6-hydroxydopamine also inhibited an increase in MAPC activity. Propranolol, trimethaphan camsylate, or hexamethonium had no effect on DCA stimulation of MAPC activity. Although the concentration of bile salt increased in the mesenteric venous outflow from the colonic loop, the intravenous administration of bile salt did not stimulate colonic MAPC activity. These studies suggest: (a) the action of DCA on smooth muscle activity is a local phenomenon, (b) the increase in MAPC activity is dependent on intact cholinergic and alpha adrenergic neurons, and (c) an increase in the concentration of bile salts in the serum is not associated with an increase in colonic MAPC activity.     

Failure of spermatogenesis in mouse lines deficient in the Na(+)-K(+)-2Cl(-) cotransporter

The Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) carries 1 molecule of Na(+) and K(+) along with 2 molecules of Cl(-) across the cell membrane. It is expressed in a broad spectrum of tissues and has been implicated in cell volume regulation and in ion transport by secretory epithelial tissue. However, the specific contribution of NKCC1 to the physiology of the various organ systems is largely undefined. We have generated mouse lines carrying either of 2 mutant alleles of the Slc12a2 gene, which encodes this cotransporter: a null allele and a mutation that results in deletion of 72 amino acids of the cytoplasmic domain. Both NKCC1-deficient mouse lines show behavioral abnormalities characteristic of mice with inner ear defects. Male NKCC1-deficient mice are infertile because of defective spermatogenesis, as shown by the absence of spermatozoa in histological sections of their epididymides and the small number of spermatids in their testes. Consistent with this observation, we show that Slc12a2 is expressed in Sertoli cells, pachytene spermatocytes, and round spermatids isolated from wild-type animals. Our results indicate a critical role for NKCC1-mediated ion transport in spermatogenesis and suggest that the cytoplasmic domain of NKCC1 is essential in the normal functioning of this protein.     

Hormonal regulation of proton secretion in rabbit medullary collecting duct.

With the exception of aldosterone, little is known about the hormonal regulation of distal nephron acidification. These experiments investigated the effects of prostaglandin E2, indomethacin, lysyl-bradykinin, 8-bromo-cyclic AMP, and forskolin on proton secretion in the major acidifying segment of the distal nephron, the medullary collecting duct from inner stripe of outer medulla. Using in vitro microperfusion and microcalorimetry, net bicarbonate reabsorption (proton secretion) was measured in rabbit medullary collecting ducts before, during, and after exposure to each test substance. PGE2 reduced proton secretion 12.2%, while the following substances stimulated proton secretion: indomethacin 14.2%; 8-bromo-cyclic AMP 34.5%; forskolin 39%. Lysyl-bradykinin was without effect. These studies demonstrate that distal nephron acidification, in addition to being stimulated by aldosterone, is significantly inhibited by the hormone PGE2. The stimulation of proton secretion by cAMP suggests that other hormones known to activate adenylate cyclase may also influence distal nephron acidification.     

Collecting duct hydrogen ion secretion in the rabbit: role of potassium.

The purpose of this study was to investigate distal nephron hydrogen ion secretion in the intact animal. The rabbit was chosen as the experimental model because it produces acid urine containing little ammonium. Upon replacement of the usual rabbit diet with milk, plus administration of an acid load (10 mEq/kg), the urine pH fell consistently from very alkaline values (PH greater than 7.4) to 4.8 +/- 0.2. Despite the ability to achieve high urine-to-blood hydrogen ion concentration gradients, the U-B PCO2, an index of collecting duct hydrogen ion secretion, was virtually zero. In these studies, the urine bicarbonate and buffer concentration were comparable to those observed in dog, rat, and man in which a high U-B PCO2 gradient was achieved. The rabbits studied had low plasma potassium concentrations (less than 3 mEq/L). Since potassium deficiency has been implicated in impaired urine acidification, potassium was administered, and it resulted in an increase in collecting duct hydrogen ion secretion as evidenced by a further fall in minimum urine pH during acidemia and a prompt rise in the U-B PCO2 during alkali administration. In summary, rabbits had a very low but not absent rate of collecting duct hydrogen ion secretion. Potassium administration increased the rate of hydrogen ion secretion in a segment of the collecting duct in which hydrogen ion secretion is reflected by an increase U-B PCO2.     

Role of angiotensin II in potassium-mediated stimulation of aldosterone secretion in the dog.

Potassium is known to enhance the aldosterone-stimulating action of angiotensin II. Such a synergistic interaction of potassium with angiotensin II could represent an action by angiotensin II to potentiate potassium as a stimulus. To examine for this effect of angiotensin II on potassium, plasma aldosterone levels were measured before and after an infusion of potassium chloride (15 meq i.v.) into dogs without and with prevention of angiotensin II formation by captopril, an angiotensin converting-enzyme inhibitor. In addition, responses to potassium were measured in a group of dogs receiving angiotensin II plus captopril. After potassium infusion, control dogs showed an increase of 7.7 +/- 1.9 (SEM) ng/dl (P less than 0.001) in the level of plasma aldosterone. In contrast, captopril-treated dogs showed no change in plasma aldosterone concentration in response to potassium. When angiotensin II was administered to captopril-treated dogs responsiveness to potassium administration was restored (plasma aldosterone concentration increased by 7.4 +/- 2.1 ng/dl, P less than 0.002). ACTH stimulated aldosterone secretion despite captopril treatment (P less than 0.001), however, ACTH produced a greater increase in the plasma aldosterone concentration in controls than in captopril-treated animals. It is evident from these results that stimulation of aldosterone secretion by potassium is considerably enhanced by angiotensin II. There appears to exist an important interdependence of these stimuli in the regulation of aldosterone secretion.     

Uterine prostaglandin E secretion and uterine blood flow in the pregnant rabbit.

Studies were performed in pregnant rabbits to assess the effect of inhibition of prostaglandin synthesis on uterine blood flow. Cardiac output and uteroplacental blood flow (UPBF) were measured using radiolabeled microspheres. Prostaglandin E (PGE) concentration was measured by radioimmunoassay in the uterine vein and peripheral artery of the pregnant nephrectomized rabbit. Either meclofenamate or indomethacin 2 mg/kg were utilized to inhibit prostaglandin synthesis. Systemic arterial pressure increased from 86 mm Hg to 98 mm Hg (P less than0.0001) after prostaglandin inhibition. Cardiac output was unchanged after the inhibition of prostaglandin synthesis, 326 ml/min to 7.8 ml/min. Uterine vein PGE concentration was extremely high, 172.4 ng/ml, with concomitant peripheral arterial PGE 2.1 NG/ML. Intravenous administration of either meclofenamate or indomethacin reduced uterine vein PGE to 23 ng/ml (P less than 0.01) and arterial PGE to 1.0 ng/ml (P less than 0.05). Male and nonpregnant female rabbits had lower arterial PGE, 0.37 ng/ml (P less 0.05). Studies in non-nephrectomized pregnant animals demonstrated that uteroplacental secretion of PGE was greater than five times renal secretion. These studies demonstrate that the rabbit uteroplacental unit is a rich source of PGE and suggest that production of the vasoactive lipid may have a key role in regulating UPBF during pregnancy.     

Ricinoleic acid stimulation of active anion secretion in colonic mucosa of the rat.

Perfusion of the colon with ricinoleic acid produces fluid and electrolyte accumulation. The mechanism of these changes in water and electrolyte movement is uknown. These studies were designed to determine whether ricinoleic acid effects active ion transport across isolated rat colonic mucosa. 0.5 mM Na ricinoleate produced significant increases in potential difference (3.8 +/- 0.5 mV) and short-circuit current (Isc) (99.2 +/- 10.1 muA/cm2). The increases in Isc produced by Na ricinoleate were inhibited by both removal of bicarbonate and chloride and by the presence of theophylline. The hydroxy fatty acid also resulted in a significant decrease in net Na absorption from 4.7 +/- 0.8 to 0.1 +/- 0.7 mueq/h cm2 and reversed net Cl transport from absorption (+ 4.5 +/- 0.9) to secretion (-2.2 +/- mueq/h cm2). In parallel studies 0.5 mM Na ricinoleate increased mucosal cyclic AMP content by 58%. The concentrations of Na ricinoleate required to produce detectable and maximal increases in both Isc and cyclic AMP were the same. These results provide evidence in support of the concept that hydroxy fatty acid-induced fluid and electrolyte accumulation is driven by an active ion secretory process.     

Cyclic adenosine monophosphate-stimulated bicarbonate secretion in rabbit cortical collecting tubules.

We studied the effects of cyclic AMP (cAMP) on HCO-3 transport by rabbit cortical collecting tubules perfused in vitro. Net HCO-3 secretion was observed in tubules from NaHCO3- loaded rabbits. 8-Bromo-cAMP-stimulated net HCO-3 secretion, whereas secretion fell with time in control tubules. Both isoproterenol and vasopressin (ADH) are known to stimulate adenylate cyclase in this epithelium; however, only isoproterenol stimulated net HCO-3 secretion. The mechanism of cAMP-stimulated HCO-3 secretion was examined. If both HCO-3 and H+ secretion were to occur simultaneously in tubules exhibiting net HCO-3 secretion, cAMP might increase the net HCO-3 secretory rate by inhibiting H+ secretion, by stimulating HCO-3 secretion, or both. These possibilities were examined using basolateral addition of the disulfonic stilbene (4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS). In acidifying tubules from NH4Cl-loaded rabbits, DIDS eliminated HCO-3 reabsorption, a result consistent with known effects of DIDS as an inhibitor of H+ secretion. In contrast, cAMP left acidification (H+ secretion) intact. DIDS applied to HCO-3 secretory tubules failed to increase the HCO-3 secretory rate, indicating minimal H+ secretion in HCO-3 secreting tubules. Thus, inhibition of H+ secretion by cAMP could not account for the cAMP-induced stimulation of net HCO-3 secretion. cAMP-stimulated HCO-3 secretion was reversibly eliminated by 0 Cl perfusate, whereas luminal DIDS had no effect. Bath amiloride (1 mM) failed to eliminate cAMP-stimulated HCO-3 secretion when bath [Na+] was 145 mM or 5 mM. cAMP depolarized the transepithelial voltage. The collected fluid [HCO-3] after cAMP could be accounted for by electrical driving forces, suggesting that cAMP stimulates passive HCO-3 secretion. However, cAMP did not alter HCO-3 permeability measured under conditions expected to inhibit transcellular HCO-3 movement (0 Cl- solutions and bath DIDS). This measured HCO-3 permeability was not high enough to account, by passive diffusion, for the HCO-3 fluxes observed in Cl-containing solutions. We conclude the following: cAMP increased net HCO3- secretion by stimulating HCO3- secretion and not by inhibiting H+ secretion; this HCO3- secretion may have occurred by Cl-HCO3- exchange; Na+-H+ exchange appeared not to play a role in basolateral H+ extrusion under these conditions; and the stimulation of HCO3- secretion by isoproterenol, but not ADH, suggests the existence of separate cell cAMP pools or cellular heterogeneity in this cAMP response.     

Location of penetration and metabolic barriers to levobunolol in the corneal epithelium of the pigmented rabbit

The objective of this study was to determine which of the five or six corneal epithelial layers was rate-limiting in the corneal penetration and metabolism of levobunolol in the pigmented rabbit. Corneal penetration and metabolism were evaluated using the isolated cornea in the modified Ussing chamber. Levobunolol and its metabolite, dihydrolevobunolol, were assayed by reversed phase high-performance liquid chromatography using spectrophotometric detection. EDTA (0.1 and 0.5%) and benzalkonium chloride (0.005-0.05%) were used to disrupt the integrity of the corneal epithelial layers. EDTA, which loosened the tight junctions between the superficial corneal epithelial cells, reduced both the transcorneal flux and metabolism of levobunolol. In contrast, benzalkonium chloride, which disrupted the integrity of the outermost corneal epithelial layers, enhanced the transcorneal levobunolol flux while reducing its extent of metabolism. The extent of enhancement in transcorneal flux afforded by 0.025% benzalkonium chloride was comparable to that seen in the deepithelized cornea. Within 5 min of contact by the corneal epithelium with this preservative, the ratio of dihydrolevobunolol concentration on the endothelial to the epithelial side was reduced by two-thirds. Although direct confirmation is required, the above findings are consistent with the hypothesis that the rate-limiting layer to corneal penetration of levobunolol resides in the outermost two to three layers of the corneal epithelium, whereas the metabolic barrier resides in deeper lying regions.