Trypsin affects basal and stimulated osmotic water permeability in isolated toad skin

1. We investigated the effect of trypsin (Tryp) on basal, stimulated and fluphenazine (FPZ)-inhibited net water flow (Jw) through isolated toad skin (Bufo arenarum). 2. Epidermal Tryp (20 min) promoted an increase in basal Jw which was dose-dependent (maximal with 0.5 mg/ml) and was prevented by a Tryp inhibitor (SBTI). 3. Tryp treatment inhibited the subsequent response to substances known to act before (oxytocin, Oxy) or after cyclic AMP (cAMP) generation (theophylline). 4. Tryp-induced Jw was not additive with the maximal response to Oxy or theophylline and did not modify FPZ's inhibitory effect on stimulated Jw. 5. Dermal Tryp (0.5 mg/ml, 20 min) did not modify basal, but inhibited Oxy and isoproterenol-stimulated Jw, without altering the response to theophylline or db-cAMP. 6. Collectively, our results show a differential action for epidermal and dermal Tryp. Tryp's side-selective action enables its use as a pharmacological tool in the functional dissection of Jw across toad skin.     

Potassium dependence of sodium transport in frog skin

22Na+ and 42K+ fluxes across the basolateral membrane of the isolated epithelium of frog skin were investigated with regard to dependence on K+ in the basolateral solution. When K+ was removed from the basolateral solution (K+-free Ringer), there was a transient rise in short circuit current (Isc) that could be eliminated by pretreatment with ouabain. Concurrently, the apparent sodium efflux across the basolateral membrane (JNa*13) showed either no change or an immediate (1-2 min) small decrease (approximately equal to 10%) that was followed by a small transient increase. K+ fluxes showed either no change or a small decrease under these conditions. JNa*13 was partially ouabain sensitive during all of the above treatments. Furosemide partially inhibited both sodium and potassium flux after K+-free treatment. The pump, as defined by ouabain sensitivity of Na+ flux, continued to work even after 20 minutes of K+-free treatment. Pump activity may be maintained by potassium leaking from the cells that is recycled by the pump. However, the ouabain-sensitive transient rise in Isc after K+-free treatment cannot readily be explained by changes in either Na+ or K+ flux. A change in pump coupling ratio provides one explanation for these data.     

Determination of adenosine 3',5'-cyclic monophosphate levels in tissues of the free living nematode, Panagrellus redivivus.

1. Tissue levels of adenosine 3', 5'-cyclic monophosphate (c-AMP) were determined by a protein binding assay in mixed populations of the free living nematode Panagrellus redivivus. 2. The values were 2.6 pmoles/mg dry weight (range 1.3-3.4 pmoles). 3. The identity of the c-AMP assayed was confirmed by both anion exchange and TLC as well as enzymatic degradation.     

Role of antidiuretic activity in the inhibition of renin secretion by vasopressin in anesthetized dogs

The nature of the activity of vasopressin which is responsible for the inhibition of renin secretion was studied by comparing the effects of vasopressin (AVP) and analogs of AVP in anesthetized water-loaded dogs. Infusion of AVP (1.0 ng/kg/min) increased mean arterial pressure (MAP) and decreased heart rate (HR) and free water clearance (CH2O). Plasma renin activity (PRA) decreased from 11.9 +/- 4.7 to 3.8 +/- 1.7 ng/ml/3 hr (p less than 0.05). A selective antidiuretic agonist, 1-deamino-8-D-arginine vasopressin (1.0 ng/kg/min), which had no effect on MAP or HR but was effective as AVP in decreasing CH2O, decreased PRA from 13.5 +/- 4.6 to 7.0 +/- 2.9 ng/ml/3 hr (p less than 0.05). Infusion of a selective vasoconstrictor agonist, 2-phenylalanine-8-ornithine oxytocin (1.0 ng/kg/min), increased MAP and decreased HR but did not decrease CH2O or PRA. A vasoconstrictor antagonist, d(CH2)5Tyr(Me)AVP (10 micrograms/kg), completely blocked the MAP and HR responses to AVP but did not block the decrease in CH2O or PRA (5.9 +/- 1.8 to 2.9 +/- 1.6 ng/ml/3 hr) (p less than 0.001). Infusion of the 0.45% saline vehicle had no significant effect on MAP, HR, CH2O or PRA. These results indicate that the inhibition of renin secretion by vasopressin in anesthetized water-loaded dogs is due to its antidiuretic activity.     

Common channels for water and protons at apical and basolateral cell membranes of frog skin and urinary bladder epithelia. Effects of oxytocin, heavy metals, and inhibitors of H(+)-adenosine triphosphatase

We have compared the response of proton and water transport to oxytocin treatment in isolated frog skin and urinary bladder epithelia to provide further insights into the nature of water flow and H+ flux across individual apical and basolateral cell membranes. In isolated spontaneous sodium-transporting frog skin epithelia, lowering the pH of the apical solution from 7.4 to 6.4, 5.5, or 4.5 produced a fall in pHi in principal cells which was completely blocked by amiloride (50 microM), indicating that apical Na+ channels are permeable to protons. When sodium transport was blocked by amiloride, the H+ permeability of the apical membranes of principal cells was negligible but increased dramatically after treatment with antidiuretic hormone (ADH). In the latter condition, lowering the pH of the apical solution caused a voltage-dependent intracellular acidification, accompanied by membrane depolarization, and an increase in membrane conductance and transepithelial current. These effects were inhibited by adding Hg2+ (100 microM) or dicyclohexylcarbodiimide (DCCD, 10(-5) M) to the apical bath. Net titratable H+ flux across frog skin was increased from 30 +/- 8 to 115 +/- 18 neq.h-1.cm-2 (n = 8) after oxytocin treatment (at apical pH 5.5 and serosal pH 7.4) and was completely inhibited by DCCD (10(-5) M). The basolateral membranes of the principal cells in frog skin epithelium were found to be spontaneously permeable to H+ and passive electrogenic H+ transport across this membrane was not affected by oxytocin. Lowering the pH of the basolateral bathing solution (pHb) produced an intracellular acidification and membrane depolarization (and an increase in conductance when the normal dominant K+ conductance of this membrane was abolished by Ba2+ 1 mM). These effects of low pHb were blocked by micromolar concentrations of heavy metals (Zn2+, Ni2+, Co2+, Cd2+, and Hg2+). Lowering pHb in the presence of oxytocin (50 mU/ml) produced a transepithelial current (3 microA.cm-2 at pHb 5.5) which was blocked by 100 microM of Hg2+, Zn2+, or Ni2+ at the basolateral side, and by DCCD (10(-5) M) or Hg2+ (100 microM) from the apical side. The net hydroosmotic water flux (JH2O) induced by oxytocin in frog bladder sacs was blocked by inhibitors of H(+)-adenosine triphosphatase (ATPase). Diethylstilbestrol (DES 10(-5) M), oligomycin (10(-8) M), and DCCD (10(-5) M) prevented JH2O when present in the lumen. These effects cannot be attributed to inhibition of metabolism since cyanide (10(-4) M), or 2-deoxyglucose (10(-3) M) had no effect on JH2O.(ABSTRACT TRUNCATED AT 400 WORDS)     

Capacitance,short-circuit current and osmotic water flow across different regions of the isolated toad skin

Summary The amphibian antidiuretic hormone, arginine vasotocin, stimulated osmotic water flow across isolated skin from the pelvic but not the pectoral skin of the toad, Bufo woodhouseii. Changes in the apical membrane capacitance were not observed for either region of the skin following treatment with arginine vasotocin when there was an osmotic gradient across the tissue. In the absence of an osmotic pressure gradient, the apical membrane capacitance of the pelvic skin increased from 2.8±0.5 to 3.3±0.6 F · cm^-2 after treatment with 5 · 10^-8 M arginine vasotocin. Under these conditions, apical membrane capacitance of the pectoral skin was 1.8±0.1 F · cm^-2 and did not change significantly after arginine vasotocin treatment. The amiloride-sensitive short-circuit current across the pelvic skin was stimulated by arginine vasotocin as was the density of channels in the apical membrane as determined by fluctuation analysis. Values for channel density in the pelvic skin also correlated with apical membrane capacitance and increased from 90 to 273 channels per m² of estimated membrane area following arginine vasotocin treatment. In the pectoral skin the stimulation of short-circuit current following arginine vasotocin treatment was small and an increase in channel density could not be demonstrated. The current through single Na⁺ channels in both regions of the skin did not different either before or after arginine vasotocin treatment.Abbreviations A amiloride - ADH antidiuretic hormone - AVT arginine vasotocin - C capacitance - C _a capacitance of apical membrane - f _c corner frequency - i single-channel current - osmotic water flow - IMP intramembrane particles - I _sc short-circuit current - amiloride-sensitive short-circuit current - M channel density - P _o probability of a channel being open - R channel receptor - R _a apical resistance - R _p paracellular resistance     

Effects of c-AMP, caffeine, theophylline, and vinblastine on spontaneous transmitter release at locust nerve-muscle junctions

The effects of c-AMP, phosphodiesterase inhibitors (caffeine and theophylline) and vinblastine on spontaneous transmitter release was investigated at locust neuromuscular junctions. c-AMP, theophylline, caffeine, and vinblastine caused facilitation of transmitter release. None of these drugs had any effect on the amplitude of miniature excitatory postsynaptic potentials (min. E.P.S.P.'s), or on the resting membrane potential, but vinblastine increased the proportion of 'large' min. E.P.S.P.'s. The effect of theophylline (but not c-AMP and caffeine) on min. E.P.S.P. frequency was found to be calcium dependent. The effects of these drugs on the locust glutamatergic synapse are compared with their actions at other synapses.     

Oxygen absorption by skin exposed to oxygen supersaturated water

The present study tests the hypothesis that skin on the plantar surface of the foot absorbs oxygen (O(2)) when immersed in water that has a high dissolved O(2) content. Healthy male and female subjects (24.2 ± 1.4 years) soaked each foot in tap water (1.7 ± 0.1 mg O(2)·L(-1); 30.7 ± 0.3 °C) or O(2)-infused water (50.2 ± 1.7 mg O(2)·L(-1); 32.1 ± 0.5 °C) for up to 30 min in 50 different experiments. Transcutaneous oximetry and near infrared spectroscopy were used to evaluate changes in skin PO(2), oxygenated haemoglobin, and cytochrome oxidase aa(3) that resulted from treatment. Compared with the tap water condition, tissue oxygenation index was 3.5% ± 1.3% higher in feet treated for 30 min with O(2)-infused water. This effect persisted after treatment, as skin PO(2) was higher in feet treated with O(2)-infused water at 2 min (237 ± 9 vs. 112 ± 5 mm HG) and 15 min (131 ± 1 vs. 87 ± 4 mm HG) post-treatment. When blood flow to the foot was occluded for 5 min, feet resting in O(2)-infused water maintained a 3-fold higher O(2) consumption rate than feet treated with tap water (9.1 ± 1.4 vs. 3.0 ± 1.0 μL·100 g(-1)·min(-1)). We estimate that skin absorbs 4.5 mL of O(2)·m(-2)·min(-1) from O(2)-infused water. Thus, skin absorbs appreciable amounts of O(2) from O(2)-infused water. This finding may prove useful and assist development of treatments targeting skin diseases with ischemic origin.     

Induction of V2 receptors in renal medulla of homozygous Brattleboro rats by arginine vasopressin

Homozygous Brattleboro rats display pronounced diabetes insipidus and when treated continuously with arginine vasopressin (AVP) acquire the ability to produce concentrated urine. In this study, the effects of continual AVP replacement on the pharmacological properties of the renal medullary V2 receptor and coupling to adenylate cyclase were examined. Osmotic minipumps that delivered AVP at four different rates were implanted into male homozygous Brattleboro rats. At the end of the 14 day treatment period, urine osmolalities were 280 +/- 24, 474 +/- 105, 1777 +/- 304 and 2202 +/- 175 mOsm/kg H2O for the 0, 31.25, 62.5 and 125 ng/hr treatment groups, respectively. Plasma AVP levels were below the level of detection for the 0 and 31.25 ng/hr treatment groups, and were 2.5 +/- 0.5 and 6.5 +/- 1.8 pg/ml for the 62.5 and 125 ng/hr treatment groups. Saturation experiments using [3H] AVP and renal medullary membranes revealed binding site concentrations of 57 +/- 9, 84 +/- 23, 164 +/- 17 and 150 +/- 18 fmol/mg protein for the 0, 31.25, 62.5 and 125 ng/hr treatment groups, respectively. AVP-stimulated cyclic AMP accumulation was enhanced in renal medullary membranes prepared from the 62.5 and 125 ng/hr treatment groups when compared to that in the 0 and 31.25 ng/hr treatment groups. From these results, it appears that circulating AVP is necessary for expression of functional V2 receptors in the homozygous Brattleboro rat renal medulla.     

Regulation of Sertoli cell cyclic adenosine 3':5' monophosphate phosphodiesterase activity by follicle stimulating hormone and dibutyrl cyclic AMP

Total phosphodiesterase activity was measured in Sertoli cell culture after exposure to isobutyl-methyl-xanthine, dibutyryl cyclic AMP and FSH. After 24 hr of incubation both FSH and dibutyryl cAMP caused a significant increase in total phosphodiesterase activity of Sertoli cell homogenates (control: 66 ± 16 pmoles/min/mg protein; FSH: 291 ± 25 pmoles/min/mg protein; dibutyryl cAMP: 630 ± 70 pmoles/min/mg protein). FSH stimulation was potentiated by isobutyl-methyl-xanthine. Both in the presence and absence of xanthine, the induction of phosphodiesterase was dependent on the FSH concentration, with maximal stimulation achieved with 0.5–1.0 μg FSH/ml. The induction of phosphodiesterase activity by hormone was abolished by cycloheximide treatment. The data suggest that FSH regulates phosphodiesterase activity via changes of cAMP levels in Sertoli cell in culture.     

Oxygen uptake capacity of the amphibious fish - Amphipnous cuchia (Ham) (Symbranchiformes, Amphipnoidae)

The O2 uptake capacity of Amphipnous cuchia has been determined in relation to standard temperature of 25 degrees C. The measurement of O2 uptake indicates nearly 75% of the oxygen demand to be met through the air breathing organs and 25% by the skin and vestigeal gill through water in a normal habitat. The total VO2 during aerial-aquatic gas exchange is 60.5 ml/kg/hr. The prevention of surfacing resulted in a lower O2 uptake rate (38.29 ml/kg/hr). During submergence, the utilisation of air sacs for extracting O2 by regular pumping of water in and out is peculiar to the fish. Under normal respiratory conditions (air + water), the slope for O2 uptake through air is 0.72, 0.23 for water and 0.57 for both air + water. The average ratio borne by the fish for aquatic/air breathing (ml/kg/min) is higher in fishes below 60 g body weight, and aquatic respiration predominates in fishes weighing less than 6.0 g.     

Role of angiotensin II and vasopressin in cisplatin-induced emesis

Cisplatin-containing chemotherapy regimens are known to produce intense nausea and vomiting. Angiotensin II (AII) and vasopressin (AVP) have been shown to have emetic properties. The role of these two peptides on cisplatin-induced vomiting was investigated in beagle dogs. Cisplatin (2 mg/kg, IV over 5 min) produced consistent emesis in all dogs after a mean latency time of 144 +/- 4 min. Serum Angiotensin Converting Enzyme (ACE) and plasma AII levels did not significantly change 3 hr after cisplatin administration (at the time of nausea and emesis) in control animals. AVP levels rose from 0.3 pg/ml to 7.5 pg/ml 3 hrs after cisplatin. Complete inhibition of ACE with enalapril (given at 3 mg/kg p.o., 3 hrs prior to cisplatin) reduced AII levels by 70%, but failed to significantly modify the increase in AVP levels (7.2 +/- 2.2 pg/ml), the latency time to emesis (149 +/- 2 min) and the number of emetic episodes induced by cisplatin. These results suggest that AII does not mediate cisplatin-induced emesis, nor does it mediate the increase in AVP observed at the time of emesis. We propose that AVP may be a good marker for nausea and emesis, and that increases in AVP may be neurally-mediated. The large increase in circulating AVP may represent a desirable water conservation response in anticipation of fluid losses induced by vomiting.     

Adenosine-3',5'-monophosphate in salivary glands of unfed and feeding female lone star ticks,Amblyomma americanum (L.)

1. Basal levels of cAMP in salivary glands of female lone star ticks were found to be about 5 pmoles/mg protein during all stages of feeding.2. Glands stimulated with 10⁻⁵M dopamine and 10⁻⁵M dopamine plus theophylline exhibited significant increases in cAMP/mg protein.3. After stimulation by 10⁻⁵ M dopamine was removed, cAMP decreased faster in glands from slowly feeding ticks (< 200 mg) than in glands of rapidly feeding ticks ( > 200 mg).     

Na+ and K+ transport at basolateral membranes of epithelial cells. III. Voltage independence of basolateral membrane Na+ efflux

Na+ efflux across basolateral membranes of isolated epithelia of frog skin was tested for voltage sensitivity. The intracellular Na+ transport pool was loaded with 24Na from the apical solution and the rate of isotope appearance in the basolateral solution (JNa23) was measured at timed intervals of 30 s. Basolateral membrane voltage was depolarized by either 50 mM K+, 5 mM Ba++, or 80 mM NH+4. Whereas within 30 s ouabain caused inhibition of JNa23, depolarization of Vb by 30-60 mV caused no significant change of JNa23. Thus, both pump-mediated and leak Na+ effluxes were voltage independent. Although the pumps are electrogenic, pump-mediated Na+ efflux is voltage independent, perhaps because of a nonlinear relationship between pump current and transmembrane voltage. Voltage independence of the leak Na+ efflux confirms a previous suggestion (Cox and Helman, 1983. American Journal of Physiology. 245:F312-F321) that basolateral membrane Na+ leak fluxes are electroneutral.     

The effect of pyridoxal-5-phosphate on the inhibition of platelet aggregation and adenosine-3'-5'-cyclic monophosphate accumulation in human platelets

Platelet adenosine-3'-5'-cyclic monophosphate (c-AMP) was determined in platelet rich plasma and washed platelets by a modification of the c-AMP protein binding assay. Studies were performed in the presence and absence of platelet aggregation inducers (adenosine diphosphate and thrombin) and inhibitors (pyridoxal-5-phosphate (PALP), prostaglandin E1 (PGE1), theophylline and papaverine). At 70% inhibitory concentration of platelet aggregation (PA) induced by papaverine or theophylline, a small but significant increase in platelet c-AMP level was found. With PALP however the inhibition of PA was not associated with a significant increase in the c-AMP level. PALP or theophylline potentiated greatly both the inhibition of PA and c-AMP accumulation induced by PGE1. Yet PALP potentiated the inhibition of PA induced by theophylline without increasing platelet c-AMP level. Our results indicate that the inhibition of PA by PALP is not mediated by c-AMP accumulation. However in the presence of PGE1, the increment in PA inhibition, is mediated by further indirect activation of the adenyl-cyclase.     

Stimulation by aldosterone of a conductive chloride pathway in toad skin

As a rule, chloride movement (JC1-) across amphibian skin is considered to be passive; this is implied in fact for preparations incubated in Ringer's fluid, since short-circuit current (Isc) is the quantitative expression of net, active sodium transport (JNa+). The nature of the Cl- pathway(s) was investigated by incubating amphibian skin (mostly Bufo marinus) with Cl- present on the epithelial side only, and after blocking JNa+ by combined treatment with ouabain and amiloride. In such conditions, JCl- was found to be equal to (reversed) Isc; furthermore, when JCl- was "translated" in terms of conductance, gCl-, the latter accounted almost quantitatively for transepithelial conductance, g1. When residual intratissue (i.e. intracellular) electronegativity was eliminated by replacing Na+ with K+, JCl- was larger but Isc and JCl- were still found to reflect each other, and gCl- again accounted for most, if not all, of g1. JCl- in the opposite direction, as a result of Cl- being present only on the dermal side, was negligible, and g1 was very low. Thus, in the absence of sodium transport, when experimental conditions are such that a net inward JCl- obtains, the anion apparently flows only through (a) conductive pathway(s). Aldosterone is probably involved in the regulation of this pathway, as JCl- was much lower when toads were maintained in dilute saline than in water or on moist peat; so was the fraction of the apical surface corresponding to mitochondria-rich cells.     

Beta-adrenergic control of the water permeability of the skin during rehydration in the toad Bufo arenarum.

1. Toads dehydrated to 80% of their standard weight (% SW) were rehydrated during 3 hr in distilled water. 2. Water permeability of the skin was positively correlated with the degree of dehydration in the range 80-100% SW. 3. Systemic administration of the beta-adrenergic agonist isoproterenol (5 mg/kg) 90 min after rehydration started (animals fully hydrated) increased skin permeability to the values observed in 80% SW dehydrated animals. 4. The administration of the beta-adrenergic blocker propranolol (5 mg/kg) 15 min before rehydration started produced a long-lasting decrease in water permeability during the 3 hr of rehydration. 5. The results are consistent with the hypothesis of a beta-adrenergic control of the water permeability of the skin during rehydration.     

β-adrenergic control of the water permeability of the skin during rehydration in the toad Bufo arenarum

1. Toads dehydrated to 80% of their standard weight (% SW) were rehydrated during 3 hr in distilled water.2. Water permeability of the skin was positively correlated with the degree of dehydration in the range 80–100% SW.3. Systemic administration of the β-adrenergic agonist isoproterenol (5 mg/kg) 90 min after rehydration started (animals fully hydrated) increased skin permeability to the values observed in 80% SW dehydrated animals.4. The administration of the β-adrenergic blocker propranolol (5 mg/kg) 15 min before rehydration started produced a long-lasting decrease in water permeability during the 3 hr of rehydration.5. The results are consistent with the hypothesis of a β-adrenergic control of the water permeability of the skin during rehydration.     

Cutaneous water evaporation--I. Its significance in heat-stressed birds

In a comparative study on avian cutaneous evaporation, two species of Phasianidae, Japanese quail Coturnix coturnix japonica and chukar partridge Alectoris chukar, and three species of Columbidae, palm dove Streptopelia senegalensis, collared turtle dove Streptopelia decaocto and rock pigeon Columbia livia, were investigated. The skin resistance to vapor diffusion (rs) and cutaneous water loss (CWL) were studied in these birds exposed to air temperatures (Ta) between 20 and 52 degrees C. The skin resistance was measured with Lambda instrument diffusive resistance meter. Skin resistance within the thermo-neutral zone varied between a minimum of 62 sec/cm in the palm dove exposed to 20 degrees C and a maximum of 309.1 sec/cm in the partridge exposed to 36 degrees C. The CWL values were 2.5 mg H2O/cm2.hr and 0.51 mg H2O/cm2.hr respectively. Maximum CWL of the quail and partridge was 1.9-2.1 mg H2O/cm2.hr, equivalent to a cooling capacity of about 17% of metabolic heat production at 45 degrees C Ta. In the palm dove, collared dove and pigeon CWL reached 6.8, 13.1 and 20.9 mg H2O/cm2.hr and rs values reached 31.2, 16.2 and 9.4 sec/cm respectively. The cooling capacity amounted to 51.5, 86.1 and 96.5% of metabolic heat during heat stress (52 degrees C). The significance of skin evaporation in body temperature regulation of heat-stressed birds is discussed.     

Desensitization by external Na of the cyclic AMP-dependent Na+/H+ antiporter in trout red blood cells

The erythrocytes of the trout, Salmo gairdneri, react to beta-adrenergic stimulation by activating a cyclic AMP-dependent and amiloride-sensitive Na+/H+ antiporter (see Borgese, F., F. Garcia-Romeu, and R. Motais, Journal of General Physiology, 1986, 87:551-566). The present study traces the kinetic behavior of the unidirectional Na fluxes after stimulation by isoproterenol. A very considerable increase (100-fold) of the unidirectional Na influx (JNa(in)) follows the addition of isoproterenol to the erythrocyte suspension. After 1.5 min, JNa(in) falls suddenly, and asymptotically diminishes toward the nonstimulated flux level. The unidirectional Na efflux (JNa(out)) proceeds according to similar kinetics. The decrease of JNa(in) and JNa(out)is not linked to either a change in the driving forces of the transported ions or a decrease of the cyclic AMP concentration but to a desensitization of the Na+/H+ antiporter. This desensitization is dependent on the external Na concentration and is not controlled by internal Na, cell swelling, or external Ca.