Influence of hyperosmotic shrinkage and β-adrenergic stimulation on red blood cell volume regulation and oxygen binding properties in rainbow trout and carp

Whole blood from rainbow trout and carp was subjected to hyperosmotic shock and subsequent beta-adrenergic stimulation (isoprenaline) at different oxygen tension ( PO(2)) and carbon dioxide tension ( PCO(2)) levels with the aim to evaluate changes in red blood cell (RBC) volume, pH and ion concentrations and their ultimate effect on blood O(2) transport characteristics. Hyperosmolality (addition of NaCl) induced RBC shrinkage, which was followed by a regulatory volume increase (RVI) that was larger at low than at high PO(2)and more complete in carp than in trout. Carp RBC showed practically full volume recovery within 140 min at low PO(2)and partial recovery at high PO(2), whereas RVI was partial under all PO(2)and PCO(2)conditions in trout. The RVI increased intracellular [Na(+)], water content, and, in carp, also pH (pHi), suggesting activation of Na(+)/H(+) exchange. In trout RBCs, activation of RVI was rapid but succeeded by deactivation. In carp RBCs, activation of Na(+) influx was slower but it continued, allowing full volume recovery. Shrinkage of the RBCs was associated with only minor decreases in blood oxygen saturation and oxygen affinity in both species. Thus, the oxygen affinity decrease expected on the basis of the increased concentration of intracellular haemoglobin and organic phosphates was small, and it appeared to some extent countered during RVI by an oxygen affinity increase via increased pHi. Addition of isoprenaline increased RBC volume and pHi and increased Hb oxygen saturation. The beta-adrenergic response was stronger at low compared to high PO(2) and at high compared to low PCO(2). The PO(2) dependency was largest in carp, whereas the PCO(2) (pH) dependency was more expressed in trout. The adrenergic response of trout RBCs was similar under isoosmotic and hyperosmotic conditions. In carp RBCs, the response was absent at high PO(2) under isoosmotic conditions, but interestingly it could be induced under hyperosmotic conditions. The data suggest that the RBC shrinkage occurring in fish moving from freshwater to seawater has minimal impact on blood O(2) binding properties.     

Evidence of alpha 1-adrenergic----protein kinase C----Na+/H+ antiporter-dependent increase in pinealocyte intracellular pH. Role in the adrenergic stimulation of cGMP accumulation

The regulation of intracellular pH (pHi) in isolated rat pinealocytes was studied using the fluorescent pH indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. Resting pHi was 7.09 when the extracellular pH (pHe) was 7.2. Treatment of pinealocytes with the physiological regulator of pineal function, norepinephrine, resulted in a concentration-dependent increase in pHi. Further analysis indicated that norepinephrine is probably acting via an alpha 1-adrenergic----[Ca2+]i----Ca2+/phospholipid- dependent protein kinase (protein kinase C) mechanism to activate the Na+/H+ antiporter, thereby causing cytoplasmic alkalization. A potential influence of cytosolic alkalization on the responsiveness of cyclic nucleotides to adrenergic agonists was also studied. Five analogs of the antiporter inhibitor amiloride reduced norepinephrine stimulation of cGMP accumulation with the same relative potency as they act on the antiporter. In contrast, although inhibitory effects of these compounds on cAMP accumulation were detectable, they occurred at 10-100-fold higher concentrations, and the relative potency of these inhibitors did not indicate they were acting via the antiporter. These findings provide evidence that 1) alpha 1-adrenergic receptor activation increases pinealocyte pHi through Ca2+----protein kinase C-dependent activation of the Na+/H+ antiporter; and 2) norepinephrine stimulation of cGMP accumulation is pHi-dependent. It would appear that alpha 1-adrenergic regulation of pHi via the Na+/H+ antiporter may be of general importance in the control of cGMP accumulation.     

Electrogenic ion transport in the intestine of the Australian common brushtail possum, <Emphasis Type="Italic">Trichosurus vulpecula</Emphasis>: indications of novel transport patterns in a marsupial

In this study, electrogenic ion transport in the intestine of the Australian common brushtail possum, Trichosurus vulpecula was investigated. In the ileum, a Na(+)-dependent, phloridzin- and amiloride-insensitive short-circuit current ( Isc) was present. Mucosal glucose stimulated a further phloridzin-sensitive, dose-dependent increase in Isc. A Na(+)-dependent, ouabain-sensitive Isc was also present in the caecum and colon. In the proximal and distal colon, amiloride (100 micro mol l(-1), mucosal) inhibited this Isc by 81+/-4% and 65+/-3%, respectively and the Ki for amiloride (approximately 1 micro mol l(-1)) was consistent with the inhibition of a classical epithelial Na(+) channel. In the caecum, 50% of the Isc was inhibited by amiloride (100 micro mol l(-1), mucosal). The amiloride-insensitive Isc in the colon was not due to electrogenic Cl(-) secretion, as serosal bumetanide (100 micro mol l(-1)) had no effect on the Isc. Furthermore, the secretagogues forskolin (10 micro mol l(-1)), carbachol (100 micro mol l(-1)) and dibutyryl-cAMP or dibutyryl-cGMP (100 micro mol l(-1)) did not stimulate electrogenic Cl(-) secretion by the colon. These results indicate that the transport properties of the hindgut of the possum differ significantly from those of eutherian mammals and may be associated with different functions of the hindgut of possums when compared to eutherian mammals.     

Characterization of a chloride current in the larval epidermis of the beetle Tenebrio molitor

Voltage-clamp analysis of single cuticle-attached epidermal cells dissected from the newly-ecdysed mealworm revealed the presence of a large inwardly-rectifying anion (i.e. outwardly-going) current. In many cells this current formed spontaneously on breaking into the cell with the patch pipette when the bath solution was isoosmotic with the pipette solution (415 mosmol/l). The current was evoked rapidly by electrical stimulation or by bathing the cells in hyposmotic saline (335 mosmol/l). The reversal potential of the activated current shifted in agreement with the Nernst prediction for Cl(-) when the transmembrane chloride gradient was altered by partially substituting bath or patch pipette Cl(-) with gluconate(-). Substitution of Na(+) with choline(+) or K(+) with TEA and Ba(+) in the bath or pipette solutions did not alter the reversal potential. Addition of 200 &mgr;mol/l cyclic AMP or 1 mmol/l cyclic GMP to the pipette solution increased the initial current strength and reduced the time taken to reach half peak amplitude from 117 sec to 49 sec and 41 sec, respectively. Cyclic AMP also raised the threshold at which the current developed under hyperosmotic conditions by about 20 mosmol/l. Addition of the Cl(-) channel blockers diphenylamine-2-carboxylic acid (200 &mgr;mmol/l) and diisothiocyanostilbene-2,2'-disulphonic acid (250 &mgr;mol/l) to the bath solution reduced the inwardly-rectifying anion current by 50%. This current was barely detectable in cells prepared from the mid-instar integument. This non-constitutive pattern of expression suggests that cellular Cl(-) efflux (and that of other anions) may be required during moult-cycle specific processes such as moulting fluid formation and cell volume regulation. As the strength of the epidermal anion current could be raised by the exogenous application of cytosolic cyclic nucleotides, the activity of the anion channels responsible for this current may normally be regulated by yet-to-be-identified hormone(s) or neuropeptide(s) acting on this tissue.     

Intracellular pH regulation in hepatocytes isolated from three teleost species.

The mechanisms of intracellular pH (pH(i)) regulation were studied in hepatocytes isolated from three species of teleost: rainbow trout (Oncorhynchus mykiss), black bullhead (Ameiurus melas) and American eel (Anguilla rostrata). Intracellular pH was monitored over time using the pH-sensitive fluorescent dye BCECF in response to acid loading under control conditions and in different experimental media containing either low Na(+) or Cl(-) concentrations, the Na(+)-H(+) exchanger blocker amiloride or the blocker of the V-type H(+)-ATPase, bafilomycin A(1). In trout and bullhead hepatocytes, recovery to an intracellular acid load occurred principally by way of a Na(+)-dependent amiloride-sensitive Na(+)-H(+) exchanger. In eel hepatocytes, the Na(+)-H(+) exchanger did not contribute to recovery to an acid load though evidence suggests that it is present on the cell membrane and participates in the maintenance of steady-state pH(i). The V-type H(+)-ATPase did not participate in recovery to an acid load in any species. A Cl(-)-HCO(3)(-) exchanger may play a role in recovery to an acid load in eel hepatocytes by switching off and retaining base that would normally be tonically extruded. Thus, it is clear that hepatocytes isolated from the three species are capable of regulating pH(i), principally by way of a Na(+)-H(+) exchanger and a Cl(-)-HCO(3)(-) exchanger, but do not exploit identical mechanisms for pH(i) recovery. J. Exp. Zool. 284:361-367, 1999.     

Exercise-induced stimulation of K(+) transport in human erythrocytes.

The hypothesis was tested that exercise-induced changes in plasma composition stimulate unidirectional K(+) transport (J(in)K) in human red blood cells (RBCs). Ten men performed two 30-s high-intensity leg-cycling tests separated by 4 min of rest. Antecubital venous blood was sampled before exercise and at the end of the second exercise bout. RBCs were separated from true exercise plasma, (42)K was added to plasma, and RBC K(+) transport was studied in vitro at 37 degrees C. In the second part of the study, blood from nine healthy men studied in vitro at 37 degrees C was used to test the hypothesis that exercise-simulated (ES) plasma stimulates net K(+) transport and J(in)K (measured using (86)Rb) in human RBCs. The J(in)K of resting RBCs added to true exercise plasma was 1,574 +/- 200 (SE) micromol. h(-1). l(-1) vs. 1,236 +/- 256 micromol. h(-1). l(-1) in true resting plasma at 2 min (controls). In true exercise and ES plasma, J(in)K was increased through activation of the ouabain-sensitive Na(+)-K(+) pump and the bumetanide-sensitive Na(+)-K(+)-2Cl(-) cotransporter. Increases in plasma osmolality and K(+), H(+), and epinephrine concentrations independently and in combination stimulated K(+) transport into human RBCs. In a third series of experiments, in which ES plasma K(+) concentration was continuously measured during the first 5 min of incubation of RBCs, a 1.6 +/- 0.3 mmol/l decrease in plasma K(+) concentration occurred during the first 2 min. It is concluded that RBCs transport K(+) at elevated rates in response to exercise-induced changes in plasma composition.     

The effects of endogenous diuretic and antidiuretic peptides and their second messengers in the Malpighian tubules of Tenebrio molitor: an electrophysiological study

The Malpighian tubules of Tenebrio molitor provide a model system for interpreting the actions of endogenous diuretic and antidiuretic peptides. The effects of diuretic (Tenmo-DH(37)) and antidiuretic (Tenmo-ADFa) peptides and their respective second messengers (cyclic AMP and cyclic GMP) on basolateral (V(bl)) and transepithelial (V(te)) potentials of Tenebrio Malpighian tubules were determined using conventional microelectrodes. In the presence of 6 mmol l(-1) Ba(2+), Tenmo-DH(37) (100 nmol l(-1)) reversibly hyperpolarized V(bl) and depolarized V(te). A similar response was seen with the addition of 1 mmol l(-1) cyclic AMP; however, the apical membrane potential (V(ap)) then showed a hyperpolarization, whereas a depolarization of V(ap) was observed with Tenmo-DH(37). Bafilomycin A(1) (5 micromol l(-1)) inhibited fluid secretion of stimulated tubules and reversed the hyperpolarization of V(bl) in response to Tenmo-DH(37). In response to 100 nmol l(-1) Tenmo-ADFa or 1 mmol l(-1) cyclic GMP, V(bl) and V(te) depolarized, although cyclic GMP affected membrane potentials somewhat differently by causing an initial hyperpolarization of V(bl) and V(te). In high [K(+)]-low [Na(+)] Ringer, 1 mmol l(-1) amiloride decreased fluid secretion rates, and depolarized both V(bl) and V(te). Amiloride significantly decreased luminal pH in paired experiments, indicating the presence of a K(+)/nH(+) exchanger in tubule cells of Tenebrio. The results suggest that the endogenous factors and their second messengers stimulate/inhibit fluid secretion by acting on the apical V-ATPase, basolateral K(+) transport, and possibly Cl(-) transport.     

Oxygen binding and acid base status of the blood from the freshwater turtle, Phrynops hilarii

Oxygenation studies with the whole blood of Phrynops hilarii show a P50 of 38 torr at extracellular pH (pHe) of 7.4 which corresponds to an intracellular pH (pHi) of 7.05 at 25 degrees C. The blood CO2 Bohr effect was -0.56 when related to pHi. pHi is related to pHe by the following equation: pHi = 0.75.pHe + 1.54 (r = 0.99); pHi = 0.72. pHe + 1.72 (r = 0.96) at 10 and 25 degrees C respectively. Blood pHe, for 25 degrees C, was 7.519 +/- 0.254 (n = 6). Blood gas partial pressures were: pCO2 = 25.8 +/- 3.8 torr (n = 6); pO2 = 61.7 +/- 21.2 torr (n = 6). The major red cell phosphates, in mmole/l erythrocytes, n = 6, were: ATP (3.66 +/- 0.86); GTP (0.53 +/- 0.28); 2.3-DPG (0.32 +/- 0.12) and inorganic phosphates (2.00 +/- 0.35). The plasma inorganic ion composition, n = 6, was, in mEq/l: K+ (3.04 +/- 0.40); Na+ (148.4 +/- 12.6); Ca2+ (4.75 +/- 1.32); Cl- (106.6 +/- 5.0). Additional blood parameters of interest (n = 6) were: lactate (2.07 +/- 1.72 mM in plasma); erythrocytes/mm3 (416 X 10(3) +/- 4.6 X 10(3)); leucocytes/mm3 (44636 +/- 2618); haematocrit (%) (14.5 +/- 3.6); haemoglobin, g/dl (3.2 +/- 0.5); plasma protein g/dl (4.4 +/- 0.4); osmolarity (293 +/- 10 mOsm/l). The non-bicarbonate buffer value was -22.6 mmol/kg H2O/pH. For a constant CO2 content, delta pHe/delta t = 0.0141 +/- 0.002 (n = 18) and delta pHi/delta t = 0.0157 +/- 0.003 (n = 18).     

Dopamine-induced epithelial K(+) and Na(+) movements in the salivary ducts of Periplaneta americana

K(+)- and Na(+)-selective double-barrelled microelectrodes were used for intracellular and luminal measurements in salivary ducts of Periplaneta americana. The salivary ducts were stimulated with dopamine (10(-6) mol l(-1)). Dopamine decreased intracellular [K(+)] from 112+/-17 mmol l(-1) to 40+/-13 mmol l(-1) (n=6) and increased intracellular [Na(+)] from 22+/-19 mmol l(-1) to 92+/-4 mmol l(-1) (n=6). Luminal [K(+)] was 15+/-3 mmol l(-1) in the unstimulated salivary ducts and increased to 26+/-11 mmol l(-1) upon stimulation with dopamine (n=10). Luminal [Na(+)] was insignificantly increased from 105+/-25 mmol l(-1) to 116+/-22 mmol l(-1) (n=12) by stimulation with dopamine. The potential difference across the basolateral membrane (PD(b)) was depolarized from -65+/-6 mV to -31+/-13 mV (n=12) and the transepithelial potential difference (PD(t)) was hyperpolarized from -13+/-6 mV to -22+/-7 mV (n=22, lumen negative) upon stimulation with dopamine. The re-establishment of prestimulus values of intracellular [K(+)] and [Na(+)] and PD(b) was inhibited by basolateral addition of ouabain (10(-4) mol l(-1)). Furosemide (10(-4) mol l(-1)) in the bath inhibited the dopamine-induced increase in intracellular [Na(+)], the decrease in intracellular [K(+)] and the depolarization of PD(b). We propose a model for dopamine-stimulated ion transport in the salivary ducts involving basolateral Na(+)-K(+)-2Cl(-) cotransport and active extrusion of K(+) via the apical membrane.     

Intracellular pH regulation in cultured embryonic chick heart cells. Na(+)-dependent Cl-/HCO3- exchange

The contribution of Cl-/HCO3- exchange to intracellular pH (pHi) regulation in cultured chick heart cells was evaluated using ion-selective microelectrodes to monitor pHi, Na+ (aiNa), and Cl- (aiCl) activity. In (HCO3- + CO2)-buffered solution steady-state pHi was 7.12. Removing (HCO3- + CO2) buffer caused a SITS (0.1 mM)-sensitive alkalinization and countergradient increase in aiCl along with a transient DIDS-sensitive countergradient decrease in aiNa. SITS had no effect on the rate of pHi recovery from alkalinization. When (HCO3- + CO2) was reintroduced the cells rapidly acidified, aiNa increased, aiCl decreased, and pHi recovered. The decrease in aiCl and the pHi recovery were SITS sensitive. Cells exposed to 10 mM NH4Cl became transiently alkaline concomitant with an increase in aiCl and a decrease in aiNa. The intracellular acidification induced by NH4Cl removal was accompanied by a decrease in aiCl and an increase in aiNa that led to the recovery of pHi. In the presence of (HCO3- + CO2), addition of either amiloride (1 mM) or DIDS (1 mM) partially reduced pHi recovery, whereas application of amiloride plus DIDS completely inhibited the pHi recovery and the decrease in aiCl. Therefore, after an acid load pHi recovery is HCO3o- and Nao- dependent and DIDS sensitive (but not Ca2+o dependent). Furthermore, SITS inhibition of Na(+)-dependent Cl-/HCO3- exchange caused an increase in aiCl and a decrease in the 36Cl efflux rate constant and pHi. In (HCO3- + CO2)-free solution, amiloride completely blocked the pHi recovery from acidification that was induced by removal of NH4Cl. Thus, both Na+/H+ and Na(+)-dependent Cl-/HCO3- exchange are involved in pHi regulation from acidification. When the cells became alkaline upon removal of (HCO3- + CO2), a SITS-sensitive increase in pHi and aiCl was accompanied by a decrease of aiNa, suggesting that the HCO3- efflux, which can attenuate initial alkalinization, is via a Na(+)-dependent Cl-/HCO3- exchange. However, the mechanism involved in pHi regulation from alkalinization is yet to be established. In conclusion, in cultured chick heart cells the Na(+)-dependent Cl-/HCO3- exchange regulates pHi response to acidification and is involved in the steady-state maintenance of pHi.     

Intracellular pH regulation in ventral horn neurones cultured from embryonic rat spinal cord

Intracellular pH was measured with the pH-sensitive fluorescent probe BCECF in spinal cord neurones cultured from rat embryos. At an external pH of 7.3, the average steady-state pHi was 7.18 +/- 0.03 (SEM, n = 97) and 7.02 +/- 0.01 (n = 221) in HEPES-buffered and in bicarbonate-buffered medium, respectively. In both external media, pHi was strongly dependent on external pH (pHe). In HEPES-buffered medium, pHi recovery following an acid load induced by transient application of ammonium required external Na+ and was inhibited by amiloride, indicating the presence of a Na+/H+ exchange. Na(+)- and HCO3(-)-dependent, DIDS-sensitive alkalinizing mechanisms also contributed to pHi regulation in CO2/bicarbonate-buffered medium. The presence of an electrogenic Na(+)-HCO3- cotransporter was confirmed by the alkalinizing effect of KCl application. The fact that pHi is lower in CO2/bicarbonate- than in HEPES-buffered medium and the alkalinization observed upon suppression of external Cl- suggest that the acidifying Cl-/HCO3- transporter plays an important role in defining pHi.     

Na(+) and Cl(-) transport by the urinary bladder of the freshwater rainbow trout (Oncorhynchus mykiss)

Freshwater (FW) rainbow trout (Oncorhynchus mykiss) urinary bladders mounted in vitro under symmetrical saline conditions displayed electroneutral active absorption of Na(+) and Cl(-) from the mucosal side; the transepithelial potential (V(t)) was 0.1 mV, and the short-circuit current was less than 1 microA cm(-2). Removal of Na(+) from mucosal saline decreased Cl(-) absorption by 56% and removal of Cl(-) decreased Na(+) absorption by 69%. However, active net absorption of both Na(+) and Cl(-) was not abolished when Cl(-) or Na(+) was replaced with an impermeant ion (gluconate or choline, respectively). Under physiological conditions with artificial urine (?Na(+) = 2.12 mM, ?Cl(-) = 3.51 mM) bathing the mucosal surface and saline bathing the serosal surface, transepithelial potential (V(t)) increased to a serosal positive approximately +7.6 mV. Unidirectional influx rates of both Na(+) and Cl(-) were 10-20-fold lower but active absorption of both ions still occurred according to the Ussing flux ratio criterion. Replacement of Na(+) with choline, or Cl(-) with gluconate, in the mucosal artificial urine yielded no change in unidirectional influx of Cl(-) or Na(+), respectively. However, kinetic analyses indicated a decrease in maximum Na(+) transport rate (J(max)) of 66% with no change in affinity (K(m)) in the low Cl(-) mucosal solution relative to the control solution. Similarly, there was a 79% decrease in J(max) values for Cl(-), again with no change in K(m), in the low-Na(+) mucosal bathing. The mucosal addition of DIDS, amiloride or bumetanide (10(-4) M) had no effect on either Na(+) or Cl(-) transport, under either symmetrical saline or artificial urine/saline conditions. Addition of the three drugs simultaneously (10(-4) M), or chlorothiazide (10(-3) M), under symmetrical saline conditions also had no effect on Na(+) or Cl(-) transport rates. Cyanide (10(-3) M) addition to mucosal artificial urine caused a slowly developing decrease of Na(+) influx to 59% and Cl(-) influx to 50% in the period after drug addition. Na(+) and Cl(-) reabsorption appears to be a partially coupled process in the urinary bladder of O. mykiss; transport mechanisms are both dependent upon and independent of the other ion.     

Comparison of DMO and flow cytometric methods for measuring intracellular pH and the effect of hyperthermia on the transmembrane pH gradient

Intracellular pH (pHi) was measured in both unheated and heated cells by the distribution of the weak acid, 5,5-dimethyl-2,4-oxazolidinedione-2-14C (14C-DMO), and by the fluorescence intensity ratio (I530/I630) of the pH sensitive fluorescent dye, 2',7'-bis(carboxyethyl)-5,6-carboxy-fluorescein (BCECF), analyzed by flow cytometry (FCM). BCECF-loaded Chinese hamster ovary (CHO) cells were analyzed by FCM after they had incubated in fresh medium at 37 degrees C for 90 min, during which time a decrease in fluorescence ratio stabilized. After stabilization, the pHi determined for CHO cells by the FCM method at pHe values of 6.0-8.1 agreed-within 0.1 pH units with that determined by the 14C-DMO method. There is a pH gradient across the plasma membrane that is not affected by heat. In CHO cells, the gradient, determined by DMO and FCM, is less or greater than pHe by 0.30 and 0.15 pH units at pHe 7.4 and 6.3, respectively, and in NG108-15 cells, the gradient determined by DMO increases to 0.50 pH units at pHe 6.3. Both cells maintained their pH gradients for at least 4 h after heating, although 99.9% of the cells were reproductively dead (survival of 10(-3)) after heating at 45.5 degrees C either at the normal pHe of 7.4 or at a low pHe of 6.4-6.7.     

Temperature and pH dependence of energy balance by (31)P- and (1)H-MRS in anaerobic frog muscle

The temperature (T)-dependence of energy consumption of resting anaerobic frog gastrocnemii exposed to different, changing electrochemical gradients was assessed. To this aim, the rate of ATP resynthesis (delta approximately P/deltat) was determined by (31)P- and (1)H-MRS as the sum of the rates of PCr hydrolysis (delta[PCr]/deltat) and of anaerobic glycolysis (delta[La]/ deltat, based on a approximately P/La ratio of 1.5). The investigated T levels were 15, 20 and 25 degrees C, whereas initial extracellular pH (pHe) values were 7.9, 7.3 and 7.0, i.e. higher, equal or lower, respectively, than intracellular pH (pHi). The latter was changing with T according to the neutrality point (dpH/dT=-0.0165 pH units/ degrees C). Both rates of PCr hydrolysis and of lactate accumulation and that of their sum, expressed as delta approximately P/deltat, were highly T-dependent. By contrast, the pHe-dependence of the muscle energy balance was nil or extremely limited at 15 and 20 degrees C, respectively, but remarkable at 25 degrees C (with a depression of the ATP resynthesis rate up to 25% with a decrease of pHe from 7.9 to 7.0). The pHe-dependent reduction of metabolic rate was associated with a down-regulation of anaerobic glycolysis due to reduced activity of ion-transporters controlling acid-base balance and/or to a shift from Na(+)/H(+) to a more efficient Na(+)-dependent Cl(-)/HCO(3)(-) exchanger. Uncoupling of glycogenolysis from P-metabolite concentrations, both as function of T (>or=20 degrees C) and of pHe (相似文献   

H+ extrusion by an apical vacuolar-type H+-ATPase in rat renal proximal tubules

The activity of Na+/H(+)-exchange and H(+)-ATPase was measured in the absence of CO2/HCO3 by microfluorometry at the single cell level in rat proximal tubules (superficial S1/S2 segments) loaded with BCECF [2'7'-bis(carboxyethyl)5-6-carboxyfluorescein- acetoxymethylester]. Intracellular pH (pHi) was lowered by a NH4Cl-prepulse technique. In the absence of Na+ in the superfusion solutions, pHi recovered from the acid load by a mechanism inhibited by 0.1 microM bafilomycin A1, a specific inhibitor of a vacuolar-type H(+)-ATPase. Readdition of Na+ in the presence of bafilomycin A1 produced an immediate recovery of pHi by a mechanism sensitive to the addition of 10 microM EIPA (ethylisopropylamiloride), a specific inhibitor of Na+/H+ exchange. The transport rate of the H(+)-ATPase is about 40% of Na+/H(+)-exchange activity at a similar pHi (0.218 +/- 0.028 vs. 0.507 +/- 0.056 pH unit/min. Pre-exposure of the tubules to 30 mM fructose, 0.5 mM iodoacetate and 1 mM KCN (to deplete intracellular ATP) prevented a pHi recovery in Na(+)-free media; readdition of Na+ led to an immediate pHi recovery. Tubules pre-exposed to Cl(-)-free media for 2 hr also reduced the rate of Na(+)-independent pHi recovery. In free-flow electrophoretic separations of brush border membranes and basolateral membranes, a bafilomycin A1-sensitive ATPase activity was found to be associated with the brush border membrane fraction; half maximal inhibition is at 6 x 10(-10) M bafilomycin A1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Blood sampling techniques and storage duration: effects on the presence and magnitude of the red blood cell beta-adrenergic response in rainbow trout (Oncorhynchus mykiss)

Many teleostean fish, including rainbow trout, regulate red blood cell (RBC) pH (pH(i)) in the presence of a stress-induced acidosis such as hypoxia, hypercapnia, or exhaustive exercise. This is accomplished through activation of RBC Na+/H+ exchange (beta-NHE), ultimately minimizing impairment to oxygen transport. Presence and characterization of the RBC beta-NHE in fish is best tested in blood from cannulated, resting animals; however, several studies have used blood from stressed animals drawn from the caudal vein and stored prior to use. The effects of sampling procedures and storage on the beta-NHE response is not known and is the focus of this study. Whole blood drawn from cannulated, resting rainbow trout was compared with RBCs obtained from the caudal vein rinsed and stored at 4 degrees C for 0, 6, 24, 48, 96 or 144 h. Isoproterenol (10(-5) M), a beta-adrenergic agonist, was added to hypoxia/hypercapnia incubated RBCs in vitro. In all treatments, isoproterenol induced a large beta-NHE response, and storage duration (< or =96 h) had a minimal affect, indicating that rinsing and storing is an easy and viable means by which to obtain RBCs and investigate function. Storage for 144 h still resulted in a significant RBC beta-NHE response; however, viability of RBCs may be compromised.     

Protein kinase C activating phorbolesters enhance the cyclic AMP response to parathyroid hormone,forskolin and choleratoxin in mouse calvarial bones and rat osteosarcoma cells

The protein kinase C-(PKC) activating phorbol esters 12-O-tetradecanoylphorbol-13-acetate (TPA; 100 nmol/l) and phorbol 12, 13-dibutyrate (PDBU; 100 nmol/l) enhanced basal cyclin AMP accumulation in cultured neonatal mouse calvaria. The cyclic AMP response to parathyroid hormone (PTH; 10 nmol/l) and the adenylate cyclase activators forskolin (1–3 mol/l) and choleratoxin (0.1 mg/ml) was potentiated in a more than additive manner by TPA and PDBU. In contrast, phorbol 13-monoacetate (phorb-13; 100 nmol/l), a related compound but inactive on PKC, had no effect on basal or stimulated cyclic AMP accumulation. In the presence of indomethacin (1mol/l), TPA and PDBU had no effect on cyclic AMP accumulation in calvarial bones per se, but were still able to cause a significant enhancement of the response to PTH, forskolin and choleratoxin. PTH-, forskolin- and choleratoxin-stimulated cyclic AMP accumulation in rat osteosarcoma cells UMR 106-01 was synergistically potentiated by TPA and PDBU, but not by phorb.-13. These data indicate that PKC enhances cyclic AMP formation and that the level of interaction may be at, or distal to, adenylate cyclase.     

Solute balance of a maize (Zea mays L.) source leaf as affected by salt treatment with special emphasis on phloem retranslocation and ion leaching

Strategies for avoiding ion accumulation in leaves of plants grown at high concentration of NaCl (100 mol m(-3)) in the rooting media, i.e. retranslocation via the phloem and leaching from the leaf surface, were quantified for fully developed leaves of maize plants cultivated hydroponically with or without salt, and with or without sprinkling (to induce leaching). Phloem sap, apoplastic fluid, xylem sap, solutes from leaf and root tissues, and the leachate were analysed for carbohydrates, amino acids, malate, and inorganic ions. In spite of a reduced growth rate Na(+) and Cl(-) concentrations in the leaf apoplast remained relatively low (about 4-5 mol m(-3)) under salt treatment. Concentrations of Na(+) and Cl(-) in the phloem sap of salt-treated maize did not exceed 12 and 32 mol m(-3), respectively, and thus remained lower than described for other species. However, phloem transport rates of these ions were higher than reported for other species. The relatively high translocation rate of ions found in maize may be due to the higher carbon translocation rate observed for C(4) plants as opposed to C(3) plants. Approximately 13-36% of the Na(+) and Cl(-) imported into the leaves through the xylem were exported by the phloem. It is concluded that phloem transport plays an important role in controlling the NaCl content of the leaf in maize. Surprisingly, leaching by artificial rain did not affect plant growth. Ion concentrations in the leachate were lower than reported for other plants but increased with NaCl treatment.     

Real-time measurements of the interactions between fluorescent speract and its sperm receptor

Lytechinus pictus sea urchin sperm express receptors for speract, a sperm-activating peptide derived from the homologous egg jelly coat. We found that the fluorescence of fluorophore-labeled, active, speract analogs is quenched upon receptor binding. This property allowed us to perform real-time measurements of speract-receptor interactions using intact sperm and to determine, for the first time, their association (k(on)) and dissociation (k(off)) rate constants. The high k(on) (2.4 x 10(7) M(-1 )s(-1)) and low k(off) (4.4 x 10(-6) s(-1) (95%) and 3.7 x 10(-4) s(-1) (5%)) can account for the sperm response to picomolar concentrations of speract. We also examined the influence of extracellular ions on speract-receptor interactions using the fluorescence quenching method described in this study. The association rate of speract to the receptor is dramatically reduced in Na(+)-free seawater (NaFSW), divalent cation-free seawater (DCFSW), and high-K(+) seawater (HKSW). In seawater speract induces an increase in intracellular pH (pHi), while it is unable to do so in either NaFSW or HKSW. To test if the lack of this pHi change causes the reduction in the speract association rate, pHi was increased with NH(4)Cl (10 mM) at the time labeled speract was added. Interestingly, this procedure completely (in HKSW) or partially (in NaFSW and DCFSW) restored the speract association rate to its receptor. These findings indicate that an increase in sperm pHi positively affects the receptor binding activity for this peptide and may partially explain the positive binding cooperativity displayed by the speract receptor.     

Evidence that Na+/H+ exchange regulates angiotensin II-stimulated diacylglycerol accumulation in vascular smooth muscle cells

Angiotensin II stimulation of vascular smooth muscle cells results in initial, rapid diacylglycerol (DG) formation from the polyphosphoinositides accompanied by intracellular acidification, as well as a more sustained DG accumulation which is accompanied by a prolonged intracellular alkalinization. To determine whether intracellular pH (pHi) modulates DG accumulation, NH4Cl and potassium acetate were used to alter pHi and DG formation was measured. NH4Cl (10 mM) increased pHi from 7.15 +/- 0.05 to 7.34 +/- 0.02 pH units and markedly enhanced the sustained (5 min), but not the initial (15 s), phase of DG formation in response to 100 nM angiotensin II (65 +/- 13% increase). Conversely, intracellular acidification with Na+-free buffer and potassium acetate (20 mM) decreased pHi to 6.93 +/- 0.08 and reduced subsequent angiotensin II-induced sustained DG formation by 82 +/- 9%. In intact cells, inhibition of angiotensin II-stimulated alkalinization by incubation in Na+-free buffer or by addition of the Na+/H+ exchange inhibitor dimethylamiloride (10 microM) decreased the ability of the cell to sustain DG formation, suggesting that active Na+/H+ exchange is necessary for continued DG formation. Thus, it seems that sustained, angiotensin II-induced diacylglycerol accumulation is regulated by intracellular alkalinization secondary to Na+/H+ exchange in cultured vascular smooth muscle cells.