Plasma catecholamine and haemodynamic responses to surgical endodontic anaesthetic protocols.

The effects of varying clinically relevant patterns of anaesthetic-vasoconstrictor combinations used for periradicular surgery on plasma concentrations of catecholamines and haemodynamic responses was studied in the canine model. Five mongrel dogs were anaesthetized with sodium pentobarbitol. A femoral cannula was inserted to measure central blood pressure and an ECG was used to monitor heart rate and any associated arrhythmias. Femoral venous blood samples were drawn before initial injection and at 3 and 10 min after injections. Plasma catecholamine concentrations were determined using high pressure liquid chromatography (HPLC). Injection protocols used three time periods, 30, 60 and 90 s, with solutions containing 1:100,000 and 1:50,000 adrenaline. No significant changes in heart rates or presence of arrhythmias were noted over the experimental protocol. Catecholamine levels in pico moles mL-1 were within the normal range at the 3-min sample level. At the 10-min sample time there was a more erratic range of concentrations, with most samples within the normal range. This may have been due to endogenous release of catecholamines in specific animals. The data identified trends in both the haemodynamic parameters and plasma catecholamine levels that can legitimately support the careful use of higher levels of a vasoconstrictor when patient profiles and surgical needs dictate.

Histological changes in the skin of Rana pipiens produced by either KCl loading or fasting.

The present study was performed to determine if either potassium loading or fasting results in histological changes in the skin. Rana pipiens were loaded with KCl and skin biopsies obtained (Group I). These biopsies were compared with biopsies from NaCl loaded frogs (Group II). In blind studies of microscopic sections, 13 of 17 biopsies of a mixture of I and II were correctly diagnosed by one observer and similarly, 14 of 17 of Group I and II were correctly diagnosed by a second observer (P = 0.0245 and 0.0063, respectively). The characteristics used to distinguish skins from KCl treated frogs versus controls treatment included: (i) an abundance of large euchromatin cells on or near the surface; (ii) changes in the basal cell layer with elongation and rotation of the nuclei; (iii) lighter cells in the spinosal layers; and (iv) sometimes the skin became thicker. The water-soluble nondialyzable material of the frog skin was extracted, and we found that it increased by 4.4 times following KCl loading (P < 0.05). However, the protein fraction was not increased by loading the frog for 3 days with NaHCO3. We conclude that potassium loading results in characteristic histological changes in the skin and that this is probably related to the ability of the skin to excrete potassium. In addition, a comparison of biopsies of skin from fed frogs with samples from frogs fasted for 40 to 49 days showed a change in the thickness of the skin. Skins of fed frogs averaged 57.0 +/- 1.4 mu thick compared with fasted, 39.9 +/- 2.7 mu (P < 0.001).

Metabolic acidosis, prostaglandin E2 and insulin stimulates intracellular free calcium ([Ca2+]i) in isolated cells of toad urinary bladder.

It is well known that metabolic acidosis (MA), PGE2, and insulin stimulate H+ excretion in toad urinary bladder. In addition, PGE2 has been shown to increase in the toad bladder during MA. Our present experimental findings indicate that MA, PGE2 and insulin increase [Ca2+]i and this then may be the signal for stimulation of H+ excretion in this tissue. Isolated cells of the toad urinary bladder, obtained from toads in a chronic metabolic acidosis (MA) have a significantly higher intracellular Ca2+ ([Ca2+]i) than similar cells obtained from toads in normal acid-base balance. Prostaglandin E2 (PGE2) (10(-5) M) was found to stimulate [Ca2+]i in the same normal toad bladder cells, as determined by the fluorescence ratio technique using FURA 2/AM (P < 0.05). Insulin (100 mU/ml) was also found to stimulate [Ca2+]i in toad bladder cells (P < 0.01). The increase in [Ca2+]i following PGE2 stimulation was not dependent on extracellular Ca2+, whereas the increase seen following insulin stimulation was dependent on extracellular Ca2+.

Stimulation of phosphoinositides by agents that stimulate proton secretion in toad urinary bladder.

The urinary bladder of Bufo marinus excretes H+ and this excretion is increased by metabolic acidosis (MA), insulin (IN), prostaglandin E2 (PGE2), increases CO2, and aldosterone. The purpose of this experiment was to determine whether MA, IN, PGE2, CO2, and aldosterone stimulate inositol phosphate's (IP) formation in isolated cells of toad urinary bladder. Cells were prepared by treating bladder sacs with collagenase. Cells were obtained from 10 toads in MA and 10 normal toads, suspended in 2 ml of Ringer's solution containing LiCl (10 mM), myo-inositol (5 mM), and [3H]myo-inositol (10 microCi), and then incubated for 2 hr at 25 degrees C. Cells were homogenized and the IP fractions quantitated by column chromatography and liquid scintillation counting. The results were expressed as dpm (mu MPO4)-1 (hr)-1. The IP in MA cells was 44,202 +/- 4,646 and in normal toad cells it was 31,637 +/- 3,613 (P < 0.05). In a separate experiment, cells from 10 paired hemibladders were isolated from normal toads. The cells were treated exactly as above except there were no LiCl in the bath. LiCl was added to all baths after 2 hr and the experimental cells were challenged with IN, PGE2, increases CO2, and aldosterone for 20 min. The IP were quantitated as above. IN treatment stimulated inositol bisphosphate and inositol triphosphate (P < 0.01). PGE2 and increases CO2 also stimulated inositol triphosphate (P < 0.05). Aldosterone did not alter formation of any of the IP fractions. We conclude that MA, IN, PGE2, and increases CO2 stimulate IP formation in cells of toad urinary bladder and inositol triphosphate may be an important second messenger in mediating the response of MA, IN, PGE2, and increases CO2.

The effect of aldosterone on phospholipid and phosphoinositide metabolism in rat submandibular gland.

It is well known that aldosterone stimulates Na+ transport in epithelia, including rat submandibular salivary gland. Aldosterone affects cells by causing nuclear-directed synthesis of aldosterone-induced protein. That protein then exerts its effect by: (1) phospholipid remodelling of the cell membrane (increasing permeability to Na+); (2) stimulating the Na+ pump; or (3) increasing ATP production by mitochondria. The purpose of this study was to determine if aldosterone stimulates membrane phospholipid or phosphoinositide turnover in the rat salivary gland and duct. Paired salivary glands were removed from 12 rats and main salivary-gland excretory ducts were removed from 16 rats. One-half of each group was treated with aldosterone (10(-6) M) while being incubated in Ringer's containing [32P]-orthophosphate for 2 h at 37 degrees C. The other half (controls) received no aldosterone during incubation. Phospholipids were extracted, separated and detected by thin-layer chromatography, autoradiography, and quantified by liquid scintillation counting. Results were expressed in dis/min (microM PO4)-1(h)-1. The turnover of membrane phospholipid fractions or percentage fractions was not changed significantly by aldosterone (p > 0.10). Paired submandibular salivary glands were also removed from 12 more rats and main submandibular excretory ducts from 18 rats. Tissue was incubated in 2 ml of Ringer's solution containing [3H]-myo-inositol for 2 h at 37 degrees C. Tissue was then placed in Ringer's containing LiCl and the experimental groups were challenged with aldosterone for 20 min at 37 degrees C. The tissues were then homogenized, and the inositol phosphate fractions quantified by column chromatography and liquid scintillation counting.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of membrane phospholipid metabolism by agents that increase acidification in toad urinary bladder.

Previous reports have indicated that metabolic acidosis stimulates H+ excretion, and this excretion is accompanied by an increased turnover of phospholipids (PL) in toad urinary bladder. The purpose of this experiment was to determine if other known stimulators of H+ excretion [insulin, deoxycorticosterone acetate (DOCA), epinephrine, parathyroid hormone, and CO2] might also stimulate PL turnover in the toad urinary bladder. Quarter bladders from normal toads were removed, weighed, and then incubated with [32P]orthophosphate for 2 hr at 25 degrees C. PL were extracted, separated, and detected using thin layer chromatography and autoradiography, and quantitated by liquid scintillation counting. Results were expressed in cpm (100 mg bladder)-1 (hr)-1. One quarter bladder received insulin (100 milliunits/ml), DOCA (10(-6) M), epinephrine (50 mM), parathyroid hormone (100 micrograms/ml), or 5% CO2 during the incubation, whereas the paired quarter bladder received no treatment. Phosphatidylcholine (PC) and phosphatidylinositol turnover were increased by insulin (P less than 0.025 and less than 0.05, respectively). DOCA had no effect on PL turnover, but stimulated the percentage fraction of PC (P less than 0.05) expressed as percentage fraction of total lipids. Five percent CO2 in the bath resulted in an increased rate of turnover of the PL fractions phosphatidylinositol (P less than 0.05), and the phosphatidic acid plus phosphatidyl-serine (P less than 0.01). Epinephrine and parathyroid hormone were both without effect on PL metabolism. We conclude that insulin, DOCA, and CO2 may stimulated H+ excretion in toad bladder in part by increasing turnover of membrane PL, PC, and phosphatidylinositol, and in the case of CO2, phosphatidic acid plus phosphatidylserine as well, but not PC.

Prostaglandin regulation of H+ secretion in amphibian epithelia.

The role of prostaglandins in regulating H+ excretion in amphibian epithelia was investigated. The abdominal skin of the southern leopard frog Rana pipiens and the urinary bladder of the toad Bufo marinus were used to measure proton excretion across their mucosal surface. Prostaglandin F2 alpha (PGF2 alpha) produced a dose-dependent inhibition of H+ excretion across the frog skin. Frogs pretreated with ibuprofen (30 mg.kg-1.day-1 for 3 days) showed an enhanced proton excretion similar to that observed when frogs are placed in chronic metabolic acidosis. The number of mitochondria-rich cells, the cells responsible for proton excretion, was also increased in frog skins after chronic metabolic acidosis or ibuprofen treatment. Mezerein and the phorbol ester 4 beta-phorbol 12-myristate 13-acetate (4 beta-PMA), activators of protein kinase C (PCK), decreased H+ excretion in frog skin, whereas the inactive phorbol 4 alpha-PMA was without an effect. The inhibition of proton excretion was similar to that observed with PGF2 alpha and suggested that the effects of PGF2 alpha and activation of PKC were mediated through a common pathway. Frogs pretreated with ibuprofen not only had an enhanced proton excretion rate but also had a decrease in cytosolic PKC activity. In another amphibian tissue, the toad urinary bladder, PGE2 inhibited proton excretion at low doses but enhanced H+ excretion at higher doses. Toads maintained under chronic metabolic acidosis had enhanced proton excretion rates and also had a threefold increase in cellular PGE2 concentration, which was consistent with the observation that PGE2 enhanced proton excretion at high doses.(ABSTRACT TRUNCATED AT 250 WORDS)

Phospholipids and electrolyte transport.

Our understanding of the role of phospholipids in ion transport processes is only beginning to be appreciated. Although the role of polyphosphoinositide and its derived second messenger molecules IP3, diacylglycerol, and arachidonic acid are well studied, we are still not certain as to how changes in the lipid bilayer structure influence the status of ion channels. This review focused on those studies which show a strong correlation with ion conductance changes and the status of the membrane phospholipids. In addition, a number of observations point to a major role of lipid second messengers that activate enzymes involved in protein phosphorylations, i.e., protein kinase C, as major regulators of a variety of ion channels and transporters. Such lipid second messengers provide a cellular mechanism whereby hormones, neurotransmitters, and pharmacologic agents functionally control the ionic environment and intracellular pH of target cells. Some of these pathways still remain to be elucidated; however, an appreciation for the participation of membrane phospholipids in these actions has been presented.

Prostaglandins as mediators of acidification in the urinary bladder of Bufo marinus.

Experiments were performed to determine whether prostaglandins (PG) play a role in H+ and NH4+ excretion in the urinary bladder of Bufo marinus. Ten paired hemibladders from normal toads were mounted in chambers. One was control and the other hemibladder received PGE2 in the serosal medium (10(-5) M). H+ excretion was measured by change in pH in the mucosal fluid and reported in units of nmol (100 mg tissue)-1 (min)-1. NH4+ excretion was measured colorimetrically and reported in the same units. The control group H+ excretion was 8.4 +/- 1.67, while the experimental group was 16.3 +/- 2.64 (P less than 0.01). The NH4+ excretion in the experimental and control group was not significantly different. Bladders from toads in a 48-hr NH4+Cl acidosis (metabolic) did not demonstrate this response to PGE2 (P greater than 0.30). Toads were put in metabolic acidosis by gavaging with 10 ml of 120 mM NH4+Cl 3 x day for 2 days. In another experiment, we measured levels of PG in bladders from control (N) and animals placed in metabolic acidosis (MA). Bladders were removed from the respective toad, homogenized, extracted, and PG separated using high-pressure liquid chromatography and quantified against PG standards. The results are reported in ng (mg tissue)-1. PGE2 fraction in N was 1.09 +/- 0.14 and in MA was 3.21 +/- 0.63 (P less than 0.01). PGF1 alpha, F2 alpha and I2 were not significantly different in N and MA toads. Bladders were also removed from N and MA toads, and incubated in Ringer's solution containing [3H]arachidonic acid (0.2 microCi/ml) at 25 degrees C for 2 hr. Bladders were then extracted for PG and the extracts separated by thin layer chromatography. PG were identified using standards and autoradiography, scraped from plates, and counted in a scintillation detector. The results are reported in cpm/mg tissue x hr +/- SEM. In MA toads, PG6-keto-F1 alpha = 1964 +/- 342, PGF2 alpha = 1016 +/- 228, and PGE2 = 904 +/- 188; in N animals PG6-keto-F1 alpha = 625 +/- 280, PGF2 alpha = 364 +/- 85, and PGE2 = 404 +/- 104; (P less than 0.01, less than 0.025, less than 0.05, respectively). We conclude that PGE2 may be an important mediator of H+ excretion in toad urinary bladder and that endogenous PGE2 levels are increased in response to MA.

Gingival crevicular fluid prostaglandins and gingival phospholipids in experimentally-induced periodontitis in the dog.

Prostaglandins are known to be mediators of inflammation in many tissues. Their fluctuations in gingival crevicular fluid during experimentally induced periodontitis were investigated, together with the possible role of phospholipids, which were measured in normal gingiva and gingiva associated with the chronic periodontitis. Periodontitis was induced in 5 dogs in the lower premolar quadrant; the opposite quadrant was used as a control. A small amount of inter-radicular bone was removed and then a cotton-wrapped stainless steel ligature was placed around each of 3 premolar teeth below the gingival margin to provide an inflammatory irritant. Pocket depth was measured by periodontal probe; crevicular fluid was collected on absorbent paper points. PGs were analysed by HPLC and the results expressed as ng PG/microliter crevicular fluid. Measurements were taken in both quadrants at 1, 3 and 6 weeks after placement of the ligatures; PGI2, 6 keto-F1 alpha, F2 alpha, E2, E1 and D2 were detected in the crevicular fluid. At 1 week, there was no difference in PG levels between experimental and control sides. During week 3, PGI2 and PGE2 increased in the experimental crevicular fluid [214.1 +/- 49.3 (SEM) vs control 87.0 +/- 39.7; p less than 0.05 and 350.0 +/- 115 vs 162.0 +/- 14.7; p less than 0.05, respectively]. At week 6, only PGE2 was elevated in crevicular fluid (207.7 +/- 68.1 vs 99.2 +/- 45; p less than 0.025).(ABSTRACT TRUNCATED AT 250 WORDS)

Phospholipid changes during adaptation to acidosis in urinary bladder of Bufo marinus.

The purpose of this study was to determine whether phospholipids (PL) play a role in the adaptation to metabolic acidosis by toad urinary bladder epithelium. Toads were placed in an NH4Cl acidosis for 48 hr. Quarter bladders were removed and incubated with [32P]orthophosphate or [3H]arachidonic acid for 1 hr at 25 degrees C. PL were detected by thin layer chromatography, autoradiography, and quantitated by liquid scintillation counting or fractional amounts were determined from phosphate content and expressed as counts per minute per micromolar of total phosphate or as percentage of fraction of total PL. Incorporation of [3H]arachidonic acid into urinary bladder PL was measured in acidotic and normal toads. There was a higher rate of arachidonic acid incorporation into several PL in acidotic animals. Phosphatidic acid and phosphatidylserine fraction in acidosis was 37,705 +/- 6,821 and in normal bladders was 9,254 +/- 2,652 (P less than 0.005); phosphatidylcholine fraction in acidotic toads was 80,462 +/- 16,862 and in normal bladders was 26,892 +/- 5,198 (P less than 0.025); and the phosphatidylethanolamine (PE) fraction in acidotic was 48,665 +/- 10,998 and in normal animals was 17,441 +/- 3,905 (P less than 0.025). 32P labeling revealed a higher rate of incorporation in bladders from acidotic toads compared with normal toads. In the acidotic bladders, the phosphatidic acid and phosphatidylserine fraction was 19,754 +/- 3,597 and in normal bladders was 12,980 +/- 1,394 (P less than 0.05) and for PE acidotic bladders was 9,129 +/- 1,304 and in normal bladders was 3,285 +/- 416 (P less than 0.001). Fractional PL (reported as percentage of fraction of total PL based on total lipid phosphorus) analysis in normal toads revealed phosphatidylinositol = 8.1 +/- 0.6% and PE = 27 +/- 1.2%, whereas for acidotic toads phosphatidylinositol = 11 +/- 0.6% and PE = 32 +/- 1.0% (P less than 0.01 for both). Aldosterone, a known stimulator of acidification, had no effect on 32P incorporation into PL fractions of the bladder. The increase in PL turnover following induction of acidosis is consistent with increased membrane synthesis or turnover during metabolic acidosis and this may reflect an increased transport of vesicular H+-ATPase into the apical membrane or the result of a proliferation of acid-secreting mitochondria-rich cells or both.

Histological changes in the skin of Rana pipiens produced by metabolic alkalosis.

The frog skin has been shown to excrete various electrolytes, the rates can be altered by varying metabolic conditions. The present study was performed to determine if metabolic alkalosis results in histological changes in the skin that are characteristic of this state. Rana pipiens were loaded with NaHCO3 and skin biopsies obtained (I). These biopsies were compared with biopsies from either control, unloaded frogs (II), or from NaCl loaded (III) frogs. In blind studies of microscopic sections, 13 of 15 biopsies of a mixture of I and II were correctly diagnosed, and similarly, 18 of 20 of I and III were correctly diagnosed (P = 0.0037, and 0.0002, respectively). The changes due to NaHCO3 treatment included; (1) an abundance of large euchromatin cells on or near the surface; (2) changes in the basal cell layer with elongation and rotation of the nuclei; (3) lighter cells in the spinosal layer; and, (4) sometimes the skin became thicker. We conclude that metabolic alkalosis results in characteristic histological changes in the skin, and that this is probably related to the ability of the skin to excrete bicarbonate.

Insulin-mediated H+ and NH+4 excretion in the urinary bladder of Bufo marinus.

This study was done to determine if insulin mediates H+ and NH+4 excretion in the urinary bladder of Bufo marinus. Acidosis was induced by gavaging with 10 ml of 120 mM NH4Cl 3X daily for 2 days. Hemibladders were mounted between Lucite chambers. Insulin (porcine) was added to the serosal solution of the experimental bladder (10(2) mU/ml). After a 15-min equilibration the flux was measured for 2 hr. H+ excretion was measured from change in pH of the mucosal fluid and the NH+4 measured colorimetrically. The excretion was normalized for weight of bladder and reported in units of nanomoles (100 mg bladder)-1(min)-1. Plasma insulin was determined by radioimmunoassay and glucose by the glucose oxidase method. In 14 control bladders H+ excretion was 8.75 +/- 1.28 and experimental was 16.35 +/- 2.50 (P less than 0.025), while NH+4 excretion in control bladder was 3.29 +/- 0.95 and experimental was 6.58 +/- 1.89 (P less than 0.01). This response was absent when the insulin was heat inactivated (P greater than 0.2 and P greater than 0.3 respectively). Plasma insulin-like levels in 10 normal toads was 0.57 +/- 0.16 ngm/ml and in acidotic toads 1.25 +/- 0.16 ng/ml (P less than 0.025). Plasma glucose levels in 10 normal toads were 22.0 +/- 3.5 mg/dl and in 12 acidotic toads 17.8 +/- 0.75 mg/dl (P less than 0.025). We conclude that plasma insulin is increased in acidosis and that insulin stimulates excretion of H+ and NH+4 in the toad urinary bladder.

Role of sodium on H+ excretion in the integument of the leopard frog Rana pipiens.

1. The northern leopard frog, Rana pipiens, pipiens, in contrast to the southern leopard frog, Rana pipiens, berlandieri, did not demonstrate any significant H+ excretion across its integument even during a challenge of chronic metabolic acidosis. Likewise, no increase in the number of H+ secreting mitochondria-rich cells were observed in the northern frogs. 2. Under normal acid-base conditions in the southern frogs, H+ excretion was found to be dependent on mucosal sodium concentrations, whereas during chronic metabolic acidosis, H+ excretion was independent of mucosal sodium concentrations, but was amiloride sensitive. 3. High salinity adapted southern frogs, under normal and acidotic conditions, had enhanced H+ excretion rates as compared to the control non-salt adapted frogs. 4. Blood analyses demonstrated that significant acid-base changes were the result of systemic acidosis and not due to salt adaptations. Blood Na+ and K+ concentrations were also efficiently maintained during salt adaptations or chronic metabolic acidosis. 5. The results suggest that H+ excretion in epithelia can be influenced by the sodium transport state of the cell and the systemic acid-base profile. Models are proposed explaining these relationships.

Morphological changes in the skin of Rana pipiens in response to metabolic acidosis.

The skin of Rana pipiens excretes H+ and this excretion is increased by metabolic acidosis. The mitochondria-rich (MR) cells of the skin have been found to mediate this H+ transport. The purpose of this study was to determine if there is a change in the MR cells of the skin during metabolic acidosis and if the isolated split epithelia of frog skin maintains its capacity to excrete H+. Metabolic acidosis was induced by injecting 120 mM NH4Cl (0.025 ml/g body wt) into the dorsal lymph sac three times a day for 2 days. The frogs were sacrificed and collagenase-split skins from the abdomen of normal and metabolic acidotic frogs were mounted between 2-ml chambers. H+ fluxes into both the mucosal and serosal media were measured and reported in units of (nmol) (cm2)-1 (min)-1. An increase in H+ flux was seen on both the mucosal and serosal sides of the acidotic split skins. The isolated epithelia were fixed, postosmicated, and dehydrated in the chamber. They were then embedded in Spurr's resin and 1-micron sections were cut and stained with Paragon multiple stain. Coded slides were used to count various cell types. Sections were randomly selected and approximately 40,000 cells were counted. Four basic cell types were noted and confirmed by TEM photomicrographs; basal (B) cells, granular (G) cells, keratinized cells, and MR cells. The ratio of G + B cells:MR cells in the normal skins was 1.0:0.021. The ratio in acidotic skins was 1.0:0.34. The average percentage of cell population of MR cells in the normal skins was 2.08 + 0.18 and in acidotic skins 3.20 + 0.36 (P less than 0.005). We conclude that the split skin maintains the capacity to acidify the mucosal fluid. Additionally, during metabolic acidosis there is an increased number of MR cells in the skin and this increase may be an adaptive mechanism of the skin to excrete excess H+ during acidosis.

Acidification of the bathing fluid by the skin of Rana pipiens in metabolic acidosis.

The skin of Rana pipiens can be shown to excrete H+ in an in vitro preparation. This H+ excretion is increased by placing the frog in metabolic acidosis. In addition, H+ excretion is increased by the presence of HCO-3-CO2 on the serosal or inside surface of the skin. Removal of Na+ from the outside bathing solution of the skin has no apparent effect on H+ excretion. Ouabain inhibits H+ excretion by the skin of acidotic frogs almost completely, in the absence of exogenous CO2. In the presence of 5% CO2 ouabain inhibits H+ excretion by 50%. In the acidotic frog skin the H+ excretion was reduced by abolishing the spontaneous potential difference. While in the normal skin there was no effect. When the P.d. was clamped at -10 to -100 mV there was no effect on H+ excretion, while there was a slight depression of H+ excretion when the P.d. was clamped at +10 to +100 mV (outside to inside the skin). In the presence of 5% CO2 there was a marked depression of H+ excretion when clamped at -10 to -100 mV in the normal skin. In metabolic acidosis there was a marked stimulation when clamped at -10 to -100 mV.

Characteristics of proton excretion in normal and acidotic toad urinary bladder.

This study, performed on the urinary bladder of Bufo marinus, was to investigate the characteristics of H+ excretion in the normal and metabolic acidotic toad. Experiments were run in modified Ussing chambers in the presence and absence of exogenous CO2. Amiloride in the mucosal medium (1.5 X 10(-4) M) inhibited H+ excretion in the acidotic bladder but this inhibition was reversed in the presence of 5% CO2. Na+-free mucosal medium revealed a component of H+ excretion in the normal toad that is Na+-dependent and not reversed by 5% CO2. Acetazolamide (10(-3) M) had no effect on H+ excretion in normal toad but inhibits excretion in the acidotic toad both in the presence and absence of exogenous CO2. In both the presence and absence of exogenous CO2 and in the presence of varying pH gradients, the bladders from acidotic toads excreted H+ at a greater rate and were able to generate greater pH gradients than in normal bladders. Voltage clamp experiments revealed that H+ excretion in the acidotic toad was inhibited by a mucosal potential difference of -20 to -100 mV but this inhibition was completely reversed by 5% CO2. In the normal toad bladder H+ excretion was stimulated by a mucosal potential difference of -20 to -100 mV in both the presence and absence of exogenous CO2. Our evidence suggests the possibility of two H+ excretory mechanisms in toad urinary bladder each with its' own set of characteristics dependent on the acid-base state of the animal. Proposed models are presented for these mechanisms.

Increased Na+ excretion by the skin of Rana pipiens after NaCl loading.

In vivo the frog skin excretes sodium and the sodium excretion is increased in response to a NaCl load. The sodium excretion can be demonstrated in vitro, and the rate of excretion is greater in skin from NaCl-loaded animals than from control, non-loaded animals. Unidirectional 22Na flux experiments on paired frog skins, as well as 22Na and 24Na bidirectional flux experiments measured in vitro, confirm the above finding that net sodium excretion occurs in response to the NaCl load.