PKCdelta activation: a divergence point in the signaling of insulin and IGF-1-induced proliferation of skin keratinocytes

Insulin and insulin-like growth factor-1 (IGF-1) are members of the family of the insulin family of growth factors, which activate similar cellular downstream pathways. In this study, we analyzed the effects of insulin and IGF-1 on the proliferation of murine skin keratinocytes in an attempt to determine whether these hormones trigger the same signaling pathways. Increasing doses of insulin and IGF-1 promote keratinocyte proliferation in an additive manner. We identified downstream pathways specifically involved in insulin signaling that are known to play a role in skin physiology; these include activation of the Na+/K+ pump and protein kinase C (PKC). Insulin, but not IGF-1, stimulated Na+/K+ pump activity. Furthermore, ouabain, a specific Na+/K+ pump inhibitor, abolished the proliferative effect of insulin but not that of IGF-1. Insulin and IGF-1 also differentially regulated PKC activation. Insulin, but not IGF-1, specifically activated and translocated the PKCB isoform to the membrane fraction. There was no effect on PKC isoforms alpha, eta, epsilon, and zeta, which are expressed in skin. PKC8 overexpression increased keratinocyte proliferation and Na+/K+ pump activity to a degree similar to that induced by insulin but had no affect on IGF-1-induced proliferation. Furthermore, a dominant negative form of PKCdelta abolished the effects of insulin on both proliferation and Na+/K+ pump activity but did not abrogate induction of keratinocyte proliferation induced by other growth factors. These data indicate that though insulin or IGF-1 stimulation induce keratinocyte proliferation, only insulin action is specifically mediated via PKC8 and involves activation of the Na+/K+ pump.     

Enhanced insulin sensitivity in extrarenal potassium handling in uremic rats.

Translocation of potassium to the intracellular compartment is impaired in advanced chronic renal failure. The purpose of this study was to evaluate the role of endogenous insulin in the disposal of an oral potassium load in uremia. Experiments were done on male Sprague-Dawley rats. Chronic renal failure (CRF) was induced by 3/4 nephrectomy. The results show that the addition of oral glucose to a potassium load was more effective in the translocation of potassium to the intracellular compartment in uremic animals. Further, suppression of endogenous insulin secretion with somatostatin caused a much higher increase in plasma potassium (K) of uremic rats (1.09 +/- 0.15 mEq/liter in CRF vs. 0.28 +/- 0.03 mEq/liter in control). Experiments to assess the activity of the Na pump were done in soleus muscles derived from these animals. Although a 50% reduction of the basal Na pump activity was found in the uremic muscles, the addition of insulin 100 mU/ml caused a relatively greater stimulation of ouabain-sensitive 86Rb uptake in the uremic muscle as compared to the control tissue (203% vs. 77% increment). These data suggest a greater sensitivity to insulin action on extrarenal potassium disposal in uremia.     

2011 homer smith award: to serve and protect: classic and novel roles for na+,k+-adenosine triphosphatase

The ability of cells to maintain sharp ion gradients across their membranes is the foundation for the molecular transport and electrical excitability. Across animal species and cell types, Na(+),K(+)-adenosine triphosphatase (ATPase) is arguably the most powerful contributor to this phenomenon. By producing a steep concentration difference of sodium and potassium between the intracellular and extracellular milieu, Na(+),K(+)-ATPase in the tubules provides the driving force for renal sodium reabsorption. Pump activity is downregulated by natriuretic hormones, such as dopamine, and is upregulated by antinatriuretic hormones, such as angiotensin. In the past decade, studies have revealed a novel and surprising role: that Na(+),K(+)-ATPase is a transducer of signals from extracellular to intracellular compartments. The signaling function of Na(+),K(+)-ATPase is activated by ouabain, a mammalian steroid hormone, at far lower concentrations than those that inhibit pump activity. By promoting growth and inhibiting apoptosis, activation of Na(+),K(+)-ATPase exerts tissue-protective effects. Ouabain-stimulated Na(+),K(+)-ATPase signaling has recently shown clinical promise by protecting the malnourished embryonic kidney from adverse developmental programming. A deeper understanding of the tissue-protective role of Na(+),K(+)-ATPase signaling and the regulation of Na(+),K(+)-ATPase pumping activity is of fundamental importance for the understanding and treatment of kidney diseases and kidney-related hypertension.     

Increased synthesis and avp unresponsiveness of Na,K-ATPase in collecting duct from nephrotic rats.

Renal sodium retention is responsible for ascites and edema in nephrotic syndrome. In puromycin aminonucleoside (PAN)-induced nephrosis, sodium retention originates in part from the collecting duct, and it is associated with increased Na,K-ATPase activity in the cortical collecting duct (CCD). The aims of this study were to evaluate whether the outer medullary collecting duct (OMCD) also participates to sodium retention and to determine the mechanisms responsible for stimulation of Na,K-ATPase in CCD. PAN nephrosis increased Na,K-ATPase activity in the CCD but not in OMCD. The two-fold increase of Na,K-ATPase activity in CCD was associated with two-fold increases in the number of alpha and beta Na,K-ATPase subunits mRNA determined by quantitative RT-PCR and of the total amount of Na,K-ATPase alpha subunits estimated by Western blotting. PAN nephrosis also increased two-fold the amount of Na,K-ATPase alpha subunit at the basolateral membrane of CCD principal cells, as determined by Western blotting after biotinylation and streptavidin precipitation and by immunofluorescence. The intracellular pool of latent Na,K-ATPase units also increased in size and was no longer recruitable by vasopressin and cAMP. This unresponsiveness of the intracellular pool of Na,K-ATPase to vasopressin was not the result of any alteration of the molecular and functional expression of the vasopressin V(2) receptor/adenylyl cyclase (AC) complex. It is concluded that PAN nephrosis (1) does not alter sodium reabsorption in OMCD, (2) is associated with increased synthesis and membrane expression of Na,K-ATPase in the CCD, and (3) alters the normal trafficking of intracellular Na,K-ATPase units to the basolateral membrane.     

Purine nucleosides stimulate Na/K ATPase, and prolong survival in hemorrhagic shock

BACKGROUND: Hemorrhagic shock leads to the appearance of substances in plasma that depress Na/K ATPase activity leading to a rise in plasma potassium. Recently, we reported that adenosine can stimulate Na/K ATPase activity, lower the plasma potassium back to control and prolong survival in shocked rats. However, adenosine also caused bradycardia. We therefore searched for adenosine analogs that stimulate Na/K ATPase without the side effects of bradycardia. METHODS: Na/K ATPase activity was assessed using Rb uptake in erythrocytes. Pentobarbital anesthetized rats had their femoral artery and vein cannulated, bled to 35 mm Hg for 1 hour and resuscitated. RESULTS: We found that the purine nucleosides, inosine, guanosine, adenosine, deoxyadenosine and deoxyguanosine, stimulated Na/K ATPase in a dose-dependent manner and overcame partial inhibition by ouabain. However, the de-ribosylated bases, the nucleotides and the pyrimidines had little or no effect on Na/K ATPase activity. Purine nucleosides did not stimulate Na/K ATPase activity through adenosine receptors, as caffeine (1 mmol/L) or aminophylline (1 mmol/L) did not block stimulation. However, stimulation was blocked by inhibitors of the equilibrative nucleoside transporter (dipyridamole, 1 mmol/L, or S-(4-nitrobenzyl)-6-thioinosine, 10 micromol/L), suggesting that the mechanism of action is intracellular. Inosine, guanosine and adenosine (2.5 mmol/L) significantly increased survival of rats in hemorrhagic shock as compared with saline and cytidine controls, and lowered the shock-elevated plasma K. CONCLUSIONS: Purine nucleosides stimulate Na/K ATPase and prolong survival in hemorrhagic shock in rats, probably through an intracellular mechanism.     

Abnormalities of erythrocyte sodium transport systems in Bartter's syndrome.

The basic tubular alteration present in Bartter's syndrome is still a subject of controversy. The possibility that a generalized defect in the transmembrane ion transport underlies the disease has been extensively investigated. Previous evaluations of cellular sodium metabolism in Bartter patients showed extremely variable findings. We have examined in the red blood cells of two patients with Bartter's syndrome the intracellular Na+ and K+ concentrations, the activity of the ouabain-sensitive Na+/K+ pump, furosemide-sensitive Na+/K+ cotransport, Na+/Li+ countertransport and the rate constant of Na+ and K+ passive permeability. We have compared these values with those of healthy subjects and patients with chronic hypokalemia produced by conditions other than Bartter's syndrome. Ouabain-sensitive Na+/K+ pump activity was decreased in both patients, whereas Na+/Li+ countertransport was activated. One of the patients also exhibited markedly decreased intraerythrocyte K+ concentration and decreased furosemide-sensitive Na+/K+ cotransport. The other had increased Na+/K+ cotransport activity and Na+ passive permeability. Intracellular Na+ and passive permeability to K+ were normal in both subjects. Neither oral potassium supplementation (100 mEq/day) nor indomethacin treatment (150 mg/day) could correct these abnormalities. Our results are partially consistent with previous observations and indicate the existence of heterogenous abnormalities of erythrocyte sodium transport systems in patients with Bartter's syndrome which are not a consequence of chronic hypokalemia.     

Insulin increases sodium reabsorption in diluting segment in humans: evidence for indirect mediation through hypokalemia

To examine the mechanism of renal sodium (Na) and potassium (K) retention during insulin infusion, seven healthy volunteers underwent clearance studies without (time control) and with insulin infusion (40 mU bolus, followed by 1 mU/kg/min for 150 min). Maximal free water clearance and fractional lithium clearance (FELi) were used to analyze renal sodium handling. Insulin decreased Na excretion (from 189 +/- 25 to 121 +/- 19 mumol/min, P less than 0.01) and K excretion (from 64 +/- 8 to 19 +/- 1 mumol/min, P less than 0.01), but did not change in glomerular filtration rate. FELi increased from 29.8 +/- 1.9 to 32.3 +/- 1.9% (P less than 0.05), minimal urine osmolality decreased from 59 +/- 3 to 46 +/- 3 mOsm/kg (P less than 0.01), and the diluting segment reabsorption index increased from 88.0 +/- 0.9 to 93.7 +/- 0.9%, P less than 0.01). Insulin also decreased plasma K, from 3.91 +/- 0.08 to 3.28 +/- 0.08 mmol/liter, P less than 0.01. In a third clearance study KCl was infused simultaneously (3.75 mumol/kg/min) to prevent this fall in plasma K. In this study insulin had no effect on Na and K excretion and diluting segment reabsorption, but the rise in FELi remained. In a fourth clearance study NaCl (3.75 mumol/kg/min) instead of KCl was infused together with insulin. This maneuver did not prevent the Na and K retaining effect of insulin, nor any of its effects on renal sodium handling parameters. These data suggest that Na and K retention during insulin infusion are largely secondary to hypokalemia, which causes increased reabsorption in the diluting segment.     

Insulin restores metabolic function in cultured cortical neurons subjected to oxidative stress

We previously demonstrated that insulin has a neuroprotective role against oxidative stress, a deleterious condition associated with diabetes, ischemia, and age-related neurodegenerative diseases. In this study, we investigated the effect of insulin on neuronal glucose uptake and metabolism after oxidative stress in rat primary cortical neurons. On oxidative stress, insulin stimulates neuronal glucose uptake and subsequent metabolism into pyruvate, restoring intracellular ATP and phosphocreatine. Insulin also increases intracellular and decreases extracellular adenosine, counteracting the effect of oxidative stress. Insulin effects are apparently mediated by phosphatidylinositol 3-K and extracellular signal-regulated kinase signaling pathways. Extracellular adenosine under oxidative stress is largely inhibited after blockade of ecto-5'-nucleotidase, suggesting that extracellular adenosine results preferentially from ATP release and catabolism. Moreover, insulin appears to interfere with the ATP release induced by oxidative stress, regulating extracellular adenosine levels. In conclusion, insulin neuroprotection against oxidative stress-mediated damage involves 1) stimulation of glucose uptake and metabolism, increasing energy levels and intracellular adenosine and, ultimately, uric acid formation and 2) a decrease in extracellular adenosine, which may reduce the facilitatory activity of adenosine receptors.     

IGF-1 vs insulin: respective roles in modulating sodium transport via the PI-3 kinase/Sgk1 pathway in a cortical collecting duct cell line

Insulin and insulin-like growth factor 1 (IGF-1) may play a role in the regulation of sodium balance by increasing basal and aldosterone-stimulated transepithelial sodium transport in the aldosterone-sensitive distal nephron (ASDN). As insulin and IGF-1 are capable of binding to each other's receptor with a 50- to 100-fold lower affinity than to their cognate receptor, it is not clear which receptor mediates its respective sodium transport response in the ASDN. The aim of the present study was to characterize the IGF-1 regulation of Na(+) transport in the mCCD(cl1) cell line, a highly differentiated cell line which responds to physiological concentrations (K(1/2)=0.3 nM) of aldosterone. IGF-1 increased basal transepithelial Na(+) transport with a K(1/2) of 0.41+/-0.07 nM. Insulin dose-response curve was displaced to the right 50-fold, as compared to that of IGF-1 (K(1/2)=20.0+/-3.0 nM), indicating that it acts through the IGF type 1 receptor (IGF-1R). Co-stimulation with IGF-1 (0.3 nM) (or 30 nM insulin) and aldosterone (0.3 nM), either simultaneously or by pretreating the cells for 5 h with aldosterone, induced an additive response. The phosphatidylinositol-3' kinase (PI3-K) inhibitor LY294002 completely blocked IGF-1 and aldosterone induced and co-induced currents. As assessed by Western blotting, protein levels of the serum-, and glucocorticoid-induced kinase (Sgk1) were directly and proportionally related to the current induced by either or both IGF-1 and aldosterone, effects also blocked by the PI3-K inhibitor LY294002. IGF-1 could play an important physiological role in regulating basal sodium transport via the PI3-K/Sgk1 pathway in ASDN.     

Sodium transport in red blood cells from dialyzed uremic patients

Studies on red blood cell (RBC) sodium (Na) transport in chronic renal failure have described abnormalities in the ouabain-sensitive Na, K pump. We now report Na transport in RBC using cation flux methodology, measuring both the ouabain-sensitive Na, K pump and the ouabain-insensitive Na, K cotransport (CoT) and Na, lithium (Li) countertransport (CTT) in 28 subjects on hemodialysis, eight subjects on chronic ambulatory peritoneal dialysis (CAPD) and 29 control subjects. Intracellular cation content and passive permeability of Na were also examined. Mean Na efflux through the ouabain-sensitive Na, K pump was not reduced in dialysis patients when compared to normal subjects, whether measured in fresh cells (1.41 +/- 0.05 vs. 1.30 +/- 0.03 mmole/liter RBC/hr; P less than 0.05) or in Na-loaded cells (7.10 +/- 0.24 vs. 6.90 +/- 0.22; NS). There was, however, a marked and uniform suppression of the CoT pathway in Na-loaded cells from dialysis patients versus controls (0.14 +/- 0.02 vs. 0.41 +/- 0.05 mmole/liter RBC/hr; P less than 0.001). Mean CTT activity, as measured by Li efflux, was not different between dialysis and normal subjects. Uremic and normal RBC had similar intracellular Na or K content as well as passive permeability for either ion. This indicates that intracellular cationic homeostasis is maintained, perhaps secondary to balanced changes in cationic flux activity through these transport pathways.     

Effect of cAMP on the activity and the phosphorylation of Na+,K(+)-ATPase in rat thick ascending limb of Henle

BACKGROUND: In rat kidney medullary thick ascending limb of Henle's loop (MTAL), activation of protein kinase A (PKA) was previously reported to inhibit Na+,K(+)-ATPase activity. This is paradoxical with the known stimulatory effect of cAMP on sodium reabsorption. Because this inhibition was mediated by phospholipase A2 (PLA2) activation, a pathway stimulated by hypoxia, we evaluated the influence of oxygen supply on cAMP action on Na+,K(+)-ATPase in MTAL. METHODS: Ouabain-sensitive 86Rb uptake and Na+,K(+)-ATPase activity were measured in isolated MTALs. Cellular ATP content and the phosphorylation level of Na+,K(+)-ATPase were determined in suspensions of outer medullary tubules. Experiments were carried out under nonoxygenated or oxygenated conditions in the absence or presence of PKA activators. RESULTS: cAMP analogues or forskolin associated with 3-isobutyl-1-methylxanthine (IBMX) inhibited ouabain-sensitive 86Rb uptake in nonoxygenated MTALs. In contrast, when oxygen supply was increased, cAMP stimulated ouabain-sensitive 86Rb uptake and Na+,K(+)-ATPase activity. Improved oxygen supply was associated with increased intracellular ATP content. The phosphorylation level of the Na+,K(+)-ATPase alpha subunit was increased by cAMP analogues or forskolin associated with IBMX in oxygenated as well as in nonoxygenated tubules. Under nonoxygenated conditions, the inhibition of Na+,K(+)-ATPase was dissociated from its cAMP-dependent phosphorylation, whereas under oxygenated conditions, the stimulatory effect of cAMP analogues on ouabain-sensitive 86Rb uptake was linearly related and cosaturated with the level of phosphorylation of the Na+,K(+)-ATPase alpha subunit. CONCLUSION: In oxygenated MTALs, PKA-mediated stimulation of Na+,K(+)-ATPase likely participates in the cAMP-dependent stimulation of sodium reabsorption. Under nonoxygenated conditions, this stimulatory pathway is likely overridden by the PLA2-mediated inhibitory pathway, a possible adaptation to protect the cells against hypoxic damage.     

Role of muscle in regulating extracellular [K+

There is a positive association between diets rich in potassium, control of blood pressure, and prevention of stroke. Extracellular [K+] is regulated closely to maintain normal membrane excitability by the concerted regulatory responses of muscle and kidney. Although kidney is responsible for ultimately matching K+ output to K+ intake each day, muscle contains more than 90% of the body's K+ and can buffer changes in extracellular fluid [K+] by either acutely taking up extracellular fluid K+ or releasing intracellular fluid K+ from muscle. It long has been assumed that the changes in muscle K+ transport are a function of sodium pump (Na,K-adenosine triphosphatase [Na, K-ATPasel]) abundance, especially that of the alpha2 isoform, which predominates in skeletal muscle. To test the physiologic significance of changes in muscle Na,K-ATPase expression, we developed the K+ clamp, which measures insulin-stimulated cellular K+ uptake in vivo in the conscious rat. By using the K+ clamp we discovered that significant insulin resistance to cell K+ uptake occurs as follows: (1) early in K+ deprivation before a decrease in muscle sodium pump pool size, and (2) during glucocorticoid treatment, which increases muscle Na,K-ATPase alpha2 levels greater than 50%. We also discovered that adaptation of renal and extrarenal K+ handling to altered K+ balance often occurs without changes in plasma [K+], supporting a feedforward mechanism involving K+ sensing in the splanchnic bed and adjustment of K+ handling. These findings establish the advantage of combining molecular analyses of Na,K-ATPase expression and activity with systems analyses of cellular K+ uptake and excretion in vivo to reveal regulatory mechanisms operating to control K+ homeostasis.     

Ouabain suppresses glucose-induced mitochondrial ATP production and insulin release by generating reactive oxygen species in pancreatic islets

We examined the effects of reduced Na(+)/K(+)-ATPase activity on mitochondrial ATP production and insulin release from rat islets. Ouabain, an inhibitor of Na(+)/K(+)-ATPase, augmented 16.7 mmol/l glucose-induced insulin release in the early period but suppressed it after a delay of 20-30 min. Unexpectedly, the ATP content in an islet decreases in the presence of 16.7 mmol/l glucose when Na(+)/K(+)-ATPase activity is diminished by ouabain, despite the reduced consumption of ATP by the enzyme. Ouabain also suppressed the increment of ATP content produced by glucose even in Ca(2+)-depleted or Na(+)-depleted conditions. That mitochondrial membrane hyperpolarization and O(2) consumption in islets exposed to 16.7 mmol/l glucose were suppressed by ouabain indicates that the glycoside inhibits mitochondrial respiration but does not produce uncoupling. Ouabain induced mitochondrial reactive oxygen species (ROS) production that was blocked by myxothiazol, an inhibitor of site III of the mitochondrial respiratory chain. An antioxidant, alpha-tocopherol, also blocked ouabain-induced ROS production as well as the suppressive effect of ouabain on ATP production and insulin release. However, ouabain did not directly affect the mitochondrial ATP production originating from succinate and ADP. These results indicate that ouabain suppresses mitochondrial ATP production by generating ROS via transduction, independently of the intracellular cationic alternation that may account in part for the suppressive effect on insulin secretion.     

Deformability and electrolyte changes of erythrocytes in connection with open heart surgery

Red cell damage was studied in 16 patients undergoing open heart surgery. The damage was assessed by red cell deformability measuring red cell filtration rate (RFR) using a microfiltration technique. Simultaneous estimations of red cell sodium, potassium and water content were carried out. During and after cardiopulmonary bypass (CPB) RFR decreased with simultaneous loss of potassium and gain in sodium. These ionic changes are known to induce an increase in cellular calcium content via raised calcium influx. Since elevated intracellular calcium content provokes reduced deformability of the red cells the observed reduction in RFR in connection with CPB might at least partly be secondary to the reduction in the K+-Na+ ratio. A comparison between the regression lines between K+/Na+ and RFR for cold blood not subjected to the trauma caused by the heart-lung machine and for patient blood suggests that approximately 20% of the reduction in RFR observed in connection with CPB is due to the reduction in K+/Na+. The severity of the erythrocyte electrolyte changes was positively correlated to the amount of oxygen used in the heart-lung machine. The results suggest that the flow of oxygen and blood through the heart-lung machine should be kept as low as possible.     

Inhibition of mitochondrial Na+-Ca2+ exchanger increases mitochondrial metabolism and potentiates glucose-stimulated insulin secretion in rat pancreatic islets

The mitochondrial Na(+)-Ca(2+) exchanger (mNCE) mediates efflux of Ca(2+) from mitochondria in exchange for influx of Na(+). We show that inhibition of the mNCE enhances mitochondrial oxidative metabolism and increases glucose-stimulated insulin secretion in rat islets and INS-1 cells. The benzothiazepine CGP37157 inhibited mNCE activity in INS-1 cells (50% inhibition at IC(50) = 1.5 micro mol/l) and increased the glucose-induced rise in mitochondrial Ca(2+) ([Ca(2+)](m)) 2.1 times. Cellular ATP content was increased by 13% in INS-1 cells and by 49% in rat islets by CGP37157 (1 micro mol/l). Krebs cycle flux was also stimulated by CGP37157 when glucose was present. Insulin secretion was increased in a glucose-dependent manner by CGP37157 in both INS-1 cells and islets. In islets, CGP37157 increased insulin secretion dose dependently (half-maximal efficacy at EC(50) = 0.06 micro mol/l) at 8 mmol/l glucose and shifted the glucose dose response curve to the left. In perifused islets, mNCE inhibition had no effect on insulin secretion at 2.8 mmol/l glucose but increased insulin secretion by 46% at 11 mmol/l glucose. The effects of CGP37157 could not be attributed to interactions with the plasma membrane sodium calcium exchanger, L-type calcium channels, ATP-sensitive K(+) channels, or [Ca(2+)](m) uniporter. In hyperglycemic clamp studies of Wistar rats, CGP37157 increased plasma insulin and C-peptide levels only during the hyperglycemic phase of the study. These results illustrate the potential utility of agents that affect mitochondrial metabolism as novel insulin secretagogues.     

Hormonal and nonhormonal mechanisms of regulation of the NA,K-pump in collecting duct principal cells

In the kidney, the collecting duct (CD) is the site of final Na+ reabsorption, according to Na+ balance requirements. In this segment of the renal tubule, principal cells may reabsorb up to 5% of the filtered sodium. The driving force for this process is provided by the basolateral Na,K-adenosine triphosphatase (ATPase) (sodium pump). Na,K-ATPase activity and expression in the CD are modulated physiologically by hormones (aldosterone, vasopressin, and insulin) and nonhormonal factors including intracellular [Na+] and extracellular osmolality. In this article, we review the short- and long-term hormonal regulation of Na,K-ATPase in CD principal cells, and we analyze the integrated network of implicated signaling pathways with an emphasis on the latest findings.     

Membrane ATPase,erythrocyte sodium and potassium in haemodialysis patients

This study was undertaken to evaluate the effect of chronic renal failure as well as dialysate sodium concentration during haemodialysis on membrane ATPase activity and erythrocyte sodium and potassium concentration. Intracellular Na and K were not changed in patients when compared to normal subjects. There was, however, a significant decrease of Na-K-ATPase activity in patients versus controls. Erythrocyte sodium increased during haemodialysis with low and normal sodium dialysate. The present results suggest that sodium dialysate concentration has an influence on the intracellular cationic homeostasis.     

A kinetic study of cation transport in erythrocytes from uremic patients

We previously described in red blood cells (RBCs) from uremic patients on dialysis a reduction in sodium (Na) efflux through the Na, potassium (K) cotransport system (Na,K CoT) while Na efflux through the Na,K pump was normal. We then examined Na efflux in fresh cells and in cells loaded to obtain one level of intracellular sodium (Nai) concentration at about 25 mmol/liter cell. In the present study we used similar cation flux methodology to examine the kinetics of cation efflux through the Na,K pump and Na,K CoT in uremic patients on dialysis. RBCs were Na-loaded to attain five different levels of Nai concentration over a range of 5 to 50 mmol/liter cells using the ionophore nystatin. At each level of Na-loading, the Nai achieved was similar in RBCs from controls and patients. Ouabain-sensitive Na efflux through the Na,K pump showed no difference in rate between normals and dialysis patients. When the kinetic parameters of this transport pathway were considered, the apparent affinity (K0.5) for sodium was not significantly different between controls and patients (18.4 +/- 2.3 vs. 20.0 +/- 2.6 mmol/liter cell) and the maximal velocity of efflux (Vmax) was also not different between controls and patients (9.6 +/- 0.7 vs. 8.5 +/- 1.2 mmol/liter cell/hr). Comparison of Nai-activated Na versus K efflux rates through the Na,K CoT in normal subjects demonstrated similar saturation kinetics, (K0.5 15.8 +/- 3.3 vs. 12.2 +/- 2.8 mmol/liter cell, Vmax 0.81 +/- 0.1 vs. 0.78 +/- 0.1 mmol/liter cell/hr) consistent with the known stoichiometric ratio of 1 Na:1 K:2 Cl described for this mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of isoproterenol on spontaneous excitations in detrusor smooth muscle cells of the guinea pig.

PURPOSE: Because beta-adrenoceptor agonists would be a useful tool for the pharmacological treatment of unstable bladder, we investigated the cellular mechanisms underlying beta-adrenoceptor mediated inhibition on spontaneous excitation in detrusor smooth muscle. MATERIALS AND METHODS: Detrusor smooth muscle bundles were isolated from guinea pig bladders. Changes in membrane potential were recorded using an intracellular recording technique. In preparations loaded with the calcium indicator fura-PE3 changes in the concentration of intracellular calcium ions were measured simultaneously with membrane potential. Effects of isoproterenol on spontaneous changes in the membrane potential and intracellular Ca(2+) were examined RESULTS: Detrusor smooth muscle cells exhibited spontaneous action potentials that were associated with transient increases in intracellular Ca(2+) (calcium transients). Isoproterenol, which hyperpolarized the membrane, prevented action potentials and calcium transients. This induced inhibition of calcium transients was not affected by cyclopiazonic acid. Isoproterenol induced hyperpolarization was inhibited by inhibitors of protein kinase A, N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, hydrochloride and Rp-adenosine-3',5'-cyclic phosphorothioate. Hyperpolarization was blocked by a solution containing 30 mM. potassium but not by a range of potassium channel blockers. Ouabain and a solution of 0.5 mM. potassium also inhibited hyperpolarization. CONCLUSIONS: Our results suggest that isoproterenol prevented spontaneous action potential discharges and associated calcium transients through the activation of protein kinase A. The isoproterenol induced inhibition of intracellular Ca(2+) largely depends on the prevention of spontaneous action potentials since the contribution of the intracellular calcium store was small. Isoproterenol hyperpolarizes the membrane, probably by stimulating sodium pump activity.