Rapidly progressive hepatic alveolar echinococcosis in an ABO-incompatible renal transplant recipient.

We report on the case of an ABO-incompatible renal re-transplant recipient maintained on an intensified immunosuppressive regimen for recurrent cellular rejection episodes and transplant glomerulopathy who presented with rapidly growing hepatic tumors, radiologically suggestive of hemangiosarcoma. Upon resection and pathological work-up, the lesions revealed alveolar echinococcosis, a rare but potentially life-threatening parasitosis. Usually infection with Echinococcus multilocularis remains asymptomatic for extended periods of time and can go unrecognized for years. In the case presented, we observed an atypically rapid growth pattern of E. multilocularis that might have been due to the extent of the immunosuppressive regimen, which included repetitive anti-CD20 treatments. Retrospectively performed serological studies with enzyme-linked immunosorbent assays known to provide high sensitivity and specificity for the detection of echinococcosis in the general population, yielded ambiguous results in our immunocompromised host, which could be, in part, explained by B-cell depletion and its effects on antibody production and indirect actions on cellular immunity. In conclusion, this is the first report to our knowledge of hepatic alveolar echinococcosis in a renal transplant recipient. This case documents an altered clinical course of the parasitosis and the challenge of serological diagnostic tools under an intensified regimen of immunosuppressive agents, including rituximab.

ABO-incompatible kidney transplantation with antigen-specific immunoadsorption and rituximab - insights and uncertainties.

Several protocols have been developed to effectively overcome the blood group barrier in renal transplantation. In the evolution of these protocols, one of the latest steps was the combination of anti-CD20 treatment with antigen-specific immunoadsorptions. Over the last years we have learned that these relatively new protocols carry very promising short-term and intermediate-term results which compare favorably to the outcome of ABO-compatible living donor transplantations. Latest reports suggest that combining immunoadsorptions with rituximab does not result in an increased risk of infectious complications or tumors in the first years after transplantation compared to ABO-compatible living donor transplantations. We recently demonstrated that a majority of patients with isoagglutinin titers >1:128 can be safely transplanted using rituximab and immunoadsorptions without an added risk of early antibody-mediated rejections. We have also shown that a cost saving 'on-demand strategy' of postoperative immunoadsorptions based on careful titer monitoring can be used as an alternative to preemptively scheduled immunoadsorptions. Although rituximab and antigen-specific immunoadsorptions are significantly less invasive than splenectomy and plasma-pheresis, long-term follow-up of patients treated with a combination of anti-CD20 antibody and antigen-specific immunoadsorption will be needed to benchmark this therapeutic option in relation to more established protocols.

Nephrogenic systemic fibrosis following exposure to gadolinium-containing contrast agent.

Nephrogenic systemic fibrosis (NSF) is a disease recently described in patients with kidney failure. It is characterized by scleroderma-like thickening of the skin, subcutaneous edema and ensuing joint contractures leading to profound disability. Furthermore, involvement of internal organs has been described. Whereas the pathogenesis is not known to date, recent reports have linked NSF to high doses of gadolinium-containing contrast agents given at magnetic resonance angiography (MRA). We describe a patient with severe NSF. The patient had received erythropoietin and had undergone vascular interventions which are suspected risk factors for this disease. Notably, the disease developed shortly after the application of gadolinium at an MRA, giving support to the recently published hypothesis that gadolinium-containing contrast agents are among the causative factors. We provide a short overview and hope to raise overall awareness towards this entity and the use of MRA contrast agents in renal patients.

Paraneoplastic cerebellar degeneration and nephrotic syndrome preceding Hodgkin's disease: case report and review of the literature.

A patient presented with symptoms of cerebellar degeneration and nephrotic syndrome. A work-up at that time failed to reveal an underlying disease; however, 20 months later Hodgkin's disease was diagnosed. Hodgkin's lymphadenopathy developed 2 wk after prednisone therapy for the nephrotic syndrome had been discontinued. Systemic polychemotherapy resulted in complete remission of both Hodgkin's disease and nephrotic syndrome, while the neurological deficit persisted. Patients with unexplained cerebellar degeneration and/or nephrotic syndrome demand extensive evaluation for the presence of Hodgkin's disease, and steroid therapy may delay diagnosis.

Extracellular nucleotides regulate cellular functions of podocytes in culture.

Extracellular nucleotides are assumed to be important regulators of glomerular functions. This study characterizes purinergic receptors in podocytes. The effects of purinergic agonists on electrophysiological properties and the intracellular free Ca(2+) concentration of differentiated podocytes were examined with the patch-clamp and fura 2 fluorescence techniques. mRNA expression of purinergic receptors was investigated by RT-PCR. Purinergic agonists depolarized podocytes. Purinergic agonists similarly increased intracellular free Ca(2+) concentration of podocytes. The rank order of potency of various nucleotides on membrane voltage and free cytosolic calcium concentration was UTP approximately UDP > [adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S)] > ATP > 2-methylthioadenosine 5'-triphosphate (2-MeS-ATP) > 2'- and 3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP) > ADP-beta-S. alpha,beta-Me-ATP was without effect. In the presence of UTP, BzATP did not cause an additional depolarization of podocytes. Incubation of cells with ATP or BzATP did not induce lactate dehydrogenase release. In RT-PCR studies, mRNAs of the P2Y(1), P2Y(2), P2Y(6), and P2X(7) receptors were detected within glomeruli and podocytes. The data indicate that extracellular nucleotides modulate podocyte function mainly by an activation of both P2Y(2) and P2Y(6) receptors.

Acidic pH inhibits non-MHC-restricted killer cell functions.

Immunotherapeutic strategies in advanced stages of solid tumors have generally met with little success. Various mechanisms have been discussed permitting the escape of tumor cells from an effective antitumoral immune response. Solid tumors are known to develop regions with acidic interstitial pH. In a recent study performed in the human system, we were able to demonstrate that non-MHC-restricted cytotoxicity is inhibited by an acidic microenvironment. To get more insight into the mechanisms leading to this reduced cytotoxic activity, we have now investigated the influence of an acidic extracellular pH (pH(e)) on the killing process in detail. Unstimulated PBMC and LAK cells were used as effector cells. Both populations are able to kill tumor cells in a MHC-independent manner via perforin/granzymes or TNFalpha, whereas only IL-2-activated cells can use the killing pathway via Fas/FasL. We studied the influence of a declining pH(e) on the different killing pathways against TNFalpha-sensitive and -resistant, as well as Fas-positive and -negative, target cells. Experiments in the absence of extracellular Ca(2+) were used to discriminate the Ca(2+)-dependent perforin-mediated killing. Here we show that the release of perforin/granzyme-containing granules, the secretion of TNFalpha, and also the cytotoxic action of Fas/FasL interaction or of membrane-bound TNFalpha were considerably inhibited by declining pH(e). Furthermore, the secretion of the activating cytokine IFNgamma, as well as the release of the down-regulating cytokines IL-10 and TGF-beta(1), was strictly influenced by surrounding pH. As a pH(e) of 5.8 resulted in a nearly complete loss of cytotoxic effector cell functions without affecting their viability, we investigated the influence of pH(e) on basic cellular functions, e.g. , mitochondrial activity and regulation of intracellular pH. We found an increasing inhibition of both functions with declining pH(e). Therefore, an acidic pH(e) obviously impairs fundamental cellular regulation, which finally prevents the killing process. In summary, our data show a strict pH(e) dependence of various killer cell functions. Thus, an acidic microenvironment within solid tumors may contribute to the observed immunosuppression in vivo, compromising antitumoral defense and immunotherapy in general, respectively.

Amino acid transport in podocytes.

It has recently been shown that formation of podocyte foot processes is dependent on a constant source of lipids and proteins (Simons M, Saffrich R, Reiser J, and Mundel P. J Am Soc Nephrol 10: 1633-1639, 1999). Here we characterize amino acid transport mechanisms in differentiated cultured podocytes and investigate whether it may be disturbed during podocyte injury. RT-PCR studies detected mRNA for transporters of neutral amino acids (ASCT1, ASCT2, and B(0/+)), cationic AA (CAT1 and CAT3), and anionic AA (EAAT2 and EAAT3). Alanine (Ala), asparagine, cysteine (Cys), glutamine (Gln), glycine (Gly), leucine (Leu), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), glutamic acid (Glu), arginine (Arg), and histidine (His) depolarized podocytes and increased their whole cell conductances. Depletion of extracellular Na(+) completely inhibited the depolarization induced by Ala, Gln, Glu, Gly, Leu, and Pro and decreased the depolarization induced by Arg and His, indicating the presence of Na(+)-dependent amino acid transport. Incubation of podocytes with 100 microg/ml puromycin aminonucleoside for 24 h significantly attenuated the effects induced by the various amino acids by approximately 70%. The data indicate the existence of different amino acid transporter systems in podocytes. Alteration of amino acid transport may participate in podocyte injury and disturbed foot process formation.

Recombinant hirudin (lepirudin) as anticoagulant in intensive care patients treated with continuous hemodialysis.

BACKGROUND: Recombinant hirudin (lepirudin) is a potent direct thrombin inhibitor, which has been approved for the treatment of heparin-induced thrombocytopenia type II (HIT). Because the drug is mainly eliminated by the kidneys, a single loading dose of hirudin may induce therapeutic anticoagulation for up to one week in patients with renal insufficiency. Thus, the use of hirudin in critically ill patients with renal failure could markedly increase their bleeding risk. In this study, hirudin was used in critically ill patients with suspected HIT while on continuous venovenous hemodialysis (CVVHD). METHODS: Hirudin anticoagulation was performed in seven critically ill patients with suspected HIT. Four patients were initially anuric. Three patients had residual renal function. In all 64 CVVHD treatments (mean duration 12 hr), a polysulfone high-flux hemodialyzer (0.75 m2) with a dialysate flow rate of 1.5 liter/hr and an ultrafiltration rate of up to 200 ml/hr was used. Hirudin was given either as continuous intravenous infusion or as repetitive intravenous boli. Monitoring of anticoagulation was performed by measurements of the systemic activated partial thromboplastin time (aPTT). RESULTS: Hirudin dosage had to be individualized according to the risk of bleeding or clotting. During CVVHD, a continuous intravenous infusion (0.006 to 0.025 mg/kg body wt/hr, N = 2) or repetitive intravenous boli (0.007 to 0.04 mg/kg, N = 5) were given. Two patients required blood transfusions prior to and during hirudin treatment. In five patients without a high bleeding risk, the hirudin dose was adjusted to achieve the target aPTT (1.5 to 2.0 x baseline) in order to prevent thrombotic complications or frequent clotting in the extracorporal circuit. Hirudin dose requirements depended on residual renal function and extracorporal clearance. CONCLUSIONS: We conclude from these first clinical data that anticoagulation with hirudin in critically ill patients on continuous hemodialysis can be performed without excessive bleeding risk by combining close clinical and laboratory monitoring. The hirudin dose has to be reduced because of renal failure, and may require adjustment for residual or recovering renal function and extracorporal elimination.

Hydrogen peroxide activates ion currents in rat mesangial cells.

BACKGROUND: Hydrogen peroxide (H2O2) is an important mediator of glomerular injury, which induces proliferation and cell contraction in mesangial cells. The aim of this study was to investigate whether and which ion currents are activated during the early cellular responses to H2O2, and to study possible mechanisms of their activation. METHODS: The effect of H2O2 on membrane voltage of mesangial cells in short-term culture was investigated with the patch clamp technique in the fast whole cell configuration. RESULTS: H2O2 contracted mesangial cells and induced a concentration-dependent biphasic membrane voltage response. One hundred micromol/liter H2O2 led to a hyperpolarization of mesangial cells from -45 +/- 1 to -55 +/- 1 mV, which was followed by a sustained depolarization to -20 +/- 3 mV. The hyperpolarization induced by H2O2 was completely blocked by the K+ channel blocker Ba2+. In the presence of a low extracellular Cl- concentration (32 mmol/liter), the depolarization induced by H2O2 was significantly increased. The H2O2-induced depolarization was inhibited by 100 micromol/liter of the disulfide-reducing agent dithiothreitol, whereas higher concentrations of dithiothreitol (1 mmol/liter) were required to partially inhibit the hyperpolarization. Protein kinase C inhibitors blocked the H2O2-induced depolarization, but not the hyperpolarization. CONCLUSIONS: The data indicate that H2O2 leads to a biphasic membrane voltage response in mesangial cells: an initial transient hyperpolarization, which is due to the activation of a K+ conductance, and a subsequent depolarization, which is, at least in part, due to the activation of a Cl- conductance. The oxidation of thiol groups by H2O2 is involved in the membrane voltage response, and the depolarization may be regulated by protein kinase C.

Dopamine depolarizes podocytes via a D1-like receptor.

BACKGROUND: Dopamine influences glomerular haemodynamics and dopamine receptors have been demonstrated in the glomerulus, but little is known about the cellular effects of dopamine in glomerular cells. The aim of this study was to investigate the influence of dopamine on the cellular functions of podocytes. METHODS: The effect of dopamine on membrane voltage was investigated in differentiated mouse podocytes. The membrane voltage was measured using the patch clamp technique. Reverse transcribed-polymerase chain reaction (RT-PCR) studies were performed to investigate the expression of dopamine receptor mRNA in mouse glomeruli and podocytes. RESULTS: The addition of dopamine (100 nM-1000 microM) caused a concentration-dependent depolarization of podocytes (EC50 is approximate to 10 microM). Like dopamine, the selective agonist of the D1-like receptor, SKF 82958, depolarized podocytes in a concentration-dependent manner. (EC50 is approximate to 50 microM). SKF 82958 stimulated a time-and concentration-dependent accumulation of cyclic adenosine 3',5'-monophosphate (cAMP) in podocytes (EC50 is approximate to microM). RT-PCR studies with primers derived from mouse sequences amplified mouse mRNA for the D1-like and the D2-like receptor in glomeruli, which were obtained by the sieve technique, whereas only mRNA for the D1-like receptor was detected in cultured mouse podocytes. CONCLUSION: The data indicate that dopamine induces a cAMP-dependent depolarization via a D1-like receptor in podocytes.

Angiotensin II modulates cellular functions of podocytes.

Angiotensin II modulates cellular functions of podocytes. The aim of this study was to examine the effects of angiotensin II (Ang II) on membrane voltage (Vm) and cytosolic calcium activity ([Ca2+]i) of rat podocytes. To approach better the in vivo situation, we have developed an experimental approach that allows podocytes to be studied in the intact microdissected glomerulus. Ang II depolarized podocytes in the glomerulus (EC50 15 nM, N = 49). Like podocytes in the glomerulus, podocytes in short-term culture also depolarized in response to Ang II (10 nM, N = 5). Ang II increased [Ca2+]i in podocytes in culture (EC50 3 nM, N = 229). In a solution with reduced extracellular [Ca2+] (10 microM), Ang II-mediated [Ca2+]i increase was significantly reduced by 60% +/- 20% (N = 12). Flufenamate, an inhibitor of nonselective ion channels, inhibited Ang II-mediated increase of [Ca2+]i (IC50 20 microM, N = 29). The Ang subtype 1 (AT1) receptor antagonist losartan inhibited both Ang II-mediated depolarization and [Ca2+]i increase in podocytes (N = 5 to 35). Our results support the concept that Ang II might influence podocyte function directly via an AT1 receptor.

Angiotensin II increases the cytosolic calcium activity in rat podocytes in culture.

In the glomerulus, angiotensin II (Ang II) reduces the ultrafiltration coefficient and enhances the filtration of macromolecules. During glomerular injury, inhibition of the renin-angiotensin system by angiotensin-converting-enzyme inhibitors reduces proteinuria and retards the progression to end-stage renal insufficiency. The mechanisms by which Ang II modulates glomerular function are still a matter of investigation. To study whether Ang II may regulate the cytosolic calcium activity ([Ca2+]i) in podocytes, these cells were propagated in short-term culture and the effect of Ang II was examined with the Fura-2 microfluorescence technique in single podocytes. The cellular identity of cultured podocytes was proven by the expression of WT-1 and pp44, specific antibodies against podocytes in vivo. Ang II led to a concentration-dependent, reversible and slow increase of [Ca2+]i with an EC50 of 3 nmol/liter Ang II (N = 229). Ten nmol/liter Ang II increased [Ca2+]i from 41 +/- 9 to 260 +/- 34 nmol/liter (N = 210). In a solution with an extracellular reduced Ca2+ concentration of 10 micromol/liter, Ang II-mediated [Ca2+]i increase was significantly reduced by 60 +/- 20% (N = 12), indicating that the [Ca2+]i increase was due to a Ca2+ influx from the extracellular space and a release of Ca2+ from intracellular stores. Flufenamate, an inhibitor of non-selective ion channels, significantly inhibited Ang II-mediated increase of [Ca2+]i (IC50 = 20 micromol/liter, N = 29), whereas the L-type Ca2+ channel blocker nicardipine even in high concentrations of > 1 micromol/liter had only a small inhibitory effect. The AT1 receptor antagonist losartan inhibited Ang II-mediated [Ca2+]i increase with an IC50 of about 0.3 nmol/liter (N = 35). The data suggest that Ang II increases [Ca2+]i in podocytes by an influx of Ca2+ through non-selective channels and by a release of Ca2+ from intracellular stores. The effect of Ang II is mediated via an AT1 receptor.

Effect of intracellular pH on agonist-induced [Ca2+]i transients in HT29 cells.

In this study we examined the influence of intracellular pH (pHi) on agonist-induced changes of intracellular Ca2+ activity ([Ca2+]i) in HT29 cells. pHi and [Ca2+]i were measured microspectrofluorimetrically using BCECF and fura-2, respectively. Buffers containing trimethylamine (TriMA), NH3/NH4+ and acetate were used to clamp pHi to defined values. The magnitudes of the peak and plateau of [Ca2+]i transients induced by carbachol (CCH, 10(-6) mol/l) were greatly enhanced by an acidic pHi and nearly abolished by an alkaline pHi. The relationship between pHi and the [Ca2+]i peak was nearly linear from pHi 7.0 to 7.8. This effect of pHi was also observed at higher CCH concentrations (10(-4 )and 10(-5) mol/l), at which the inhibitory effect of an alkaline pHi was more pronounced than the stimulatory effect of an acidic pHi. An acidic pHi shifted the CCH concentration/response curve to the left, whereas an alkaline pHi led to a rightward shift. The influence of pHi on [Ca2+]i transients induced by neurotensin (10(-8) mol/l) or ATP (5 x 10(-7) mol/l) was similar to its influence on those induced by CCH, but generally not as pronounced. Measurements of cellular inositol 1,4,5-trisphosphate (InsP3) showed no changes in response to acidification with acetate (20 mmol/l) or alkalinization with TriMA (20 mmol/l). The InsP3 increase induced by CCH was unaltered at an acidic pHi, but was augmented at an alkaline pHi. Confocal measurements of cell volume showed no significant changes induced by TriMA or acetate. Slow-whole-cell patch-clamp experiments showed no additional effect of CCH on the membrane voltage (Vm) measured after TriMA or acetate application. We conclude that pHi is a physiological modulator of hormonal effects in HT29 cells, as the [Ca2+]i responses to agonists were significantly changed at already slightly altered pHi. The measurements of InsP3, cell volume and Vm show that pHi must act distally to the InsP3 production, and not via changes of cell volume or Vm.

Agonist-induced activation of a non-selective ion current in glomerular endothelial cells.

The control of intracellular calcium activity ([Ca2+]i) and membrane voltage (Vm) play an important role in regulating functions of glomerular endothelial cells (GEC). We investigated the effect of extracellular ATP on the intracellular [Ca2+]i, Vm and ion conductances in GEC. ATP (100 mumol/liter) induced a rapid increase of [Ca2+]i in GEC from 20 +/- 6 to 442 +/- 84 nmol/liter, which was followed by a sustained Ca2+ plateau of 112 +/- 29 nmol/liter. In a bath solution with a low extracellular Ca2+ concentration the ATP-induced [Ca2+]i peak was still present, but the [Ca2+]i plateau was completely prevented. In 186 experiments with the patch clamp technique the addition of ATP (1 to 100 mumol/liter) to GEC induced a transient small hyperpolarization, which was followed by a depolarization. During the ATP-induced depolarization an increase of the whole cell conductance was found. The Ca2+ ionophore A23187 (10 mumol/liter) mimicked the effect of ATP on Vm. Reduction of the extracellular Ca2+ to 1 mumol/liter itself depolarized GEC reversibly from -88 +/- 2 to -60 +/- 12 mV and increased the ATP-induced depolarization to -18 +/- 3 mV. In the absence of Na+ in the bathing solution (replacement by NMDG+) ATP induced only an attenuated depolarization and no inward current was activated. Flufenamate (100 mumol/liter), a blocker of non-selective ion channels inhibited the ATP-induced depolarization of Vm significantly by 58 +/- 13%, whereas nicardipine (10 mumol/liter) or amiloride (10 mumol/liter) had no effect. Our data indicate that the resting Vm of GEC cells is almost completely dominated by K+ conductances and that ATP activates a Ca2+ dependent non-selective ion conductance in GEC.

UTP and ATP induce different membrane voltage responses in rat mesangial cells.

UTP and ATP induce different membrane voltage responses in rat mesangial cells. Recent studies have indicated that UTP and ATP might modulate mesangial cell function in a different manner. Here we compared the effect of UTP and ATP on membrane voltage (Vm) and ion currents in mesangial cells in primary culture, and we examined whether different nucleotide receptors are involved. In patch-clamp experiments in the fast whole cell configuration, UTP (in contrast to ATP) caused a sustained and concentration-dependent depolarization (half-maximal effective dose, 10(-5) M), but ATP caused only a transient depolarization. During the depolarization, UTP induced a sustained increase of the whole cell conductance (Gm), whereas ATP induced only a transient increase of Gm. When cells were dialyzed with Cs2SO4 and extracellular Cl- was replaced by 145 mM sodium gluconate, addition of UTP or ATP (both 10(-4) M) did not significantly increase Gm. Addition of ATP in the presence of UTP caused an additional depolarization by 5 mV, which was followed by a hyperpolarization by 21 mV. Repetitive application of ATP led to an attenuation of the ATP-induced depolarization. Then, in the presence of ATP, UTP still induced a significant depolarization by 10 mV. Suramine and reactive blue 2 did not inhibit the depolarization induced by UTP, but these inhibited the Vm response to ATP. In microfluorescence experiments, UTP and ATP caused a concentration-dependent increase of the intracellular calcium activity ([Ca2+]i) in mesangial cells. Application of both UTP and ATP had no additive effect on [Ca2+]i. The results suggest that mesangial cells possess, in addition to P2y purinoceptors, separate nucleotide receptors for UTP.

Angiotensin II depolarizes podocytes in the intact glomerulus of the Rat.

The aim of this study was to examine the effects of angiotensin II (Ang II) on cellular functions of rat podocytes (pod) in the intact freshly isolated glomerulus and in culture. Membrane voltage (Vm) and ion currents of pod were examined with the patch clamp technique in fast whole cell and whole cell nystatin configuration. Vm of pod was -38+/-1 mV (n = 86). Ang II led to a concentration-dependent depolarization of pod with an ED50 of 10(-8) mol/liter. In the presence of Ang II (10(-7) mol/liter, n = 20), pod depolarized by 7+/-1 mV. In an extracellular solution with a reduced Cl- concentration of 32 mmol/liter, the effect of Ang II on Vm was significantly increased to 14+/-4 mV (n = 8). The depolarization induced by Ang II was neither inhibited in an extracellular Na+-free solution nor in a solution with a reduced extracellular Ca2+ (down to 1 micromol/liter). Like Ang II, the calcium ionophore A23187 (10(-5) mol/liter, n = 9) depolarized pod by 10+/-2 mV, whereas forskolin (10(-5) mol/liter), 8-(4-chlorophenylthio)-cAMP and N2,2'-o-dibutyryl-cGMP (both 5 x 10(-4) mol/liter) did not alter Vm of pod. The angiotensin 1 receptor antagonist losartan (10(-7) mol/liter) completely inhibited the Ang II-induced (10(-7) mol/liter) depolarization (n = 5). Like pod in the glomerulus, pod in short term culture depolarized in response to Ang II (10(-8) mol/liter, n = 5). Our results suggest that Ang II depolarizes podocytes directly by opening a Cl- conductance. The activation of this ion conductance is mediated by an AT1 receptor and may be regulated by the intracellular Ca2+ activity.

The effect of intracellular pH on cytosolic Ca2+ in HT29 cells.

The influence of intracellular pH (pHi) on intracellular Ca2+ activity ([Ca2+]i) in HT29 cells was examined microspectrofluorometrically. pHi was changed by replacing phosphate buffer by the diffusible buffers CO2/HCO3- or NH3/NH4+ (pH 7.4). CO2/HCO3- buffers at 2,5 or 10% acidified pHi by 0.1, 0.32 and 0.38 pH units, respectively, and increased [Ca2+]i by 8-15 nmol/l. This effect was independent of the extracellular Ca2+ activity and the filling state of thapsigargin-sensitive Ca2+ stores. Removing the CO2/HCO3- buffer alkalinized pHi by 0.14 (2%), 0.27 (5%), and 0.38 (10%) units and enhanced [Ca2+]i to a peak value of 20, 65, and 143 nmol/l, respectively. Experiments carried out with Ca2+-free solution and with thapsigargin showed that the [Ca2+]i transient was due to release from intracellular pools and stimulated Ca2+ entry. NH3/NH4+ (20 mmol/l) induced a transient intracellular alkalinization by 0.6 pHunits and increased [Ca2+]i to a peak (Delta [Ca2+]i = 164 nmol/l). The peak [Ca2+]i increase was not influenced by removal of external Ca2+, but the decline to basal [Ca2+]i was faster. Neither the phospholipase C inhibitor U73122 nor the inositol 1,4,5-trisphosphate (InsP3) antagonist theophylline had any influence on the NH3/NH4+-stimulated [Ca2+]i increase, whereas carbachol-induced [Ca2+]i transients were reduced by more than 80% and 30%, respectively. InsP3 measurements showed no change of InsP3 during exposure to NH3/NH4+, whereas carbachol enhanced the InsP3 concentration, and this effect was abolished by U73122. The pHi influence on "capacitative" Ca2+ influx was also examined. An acid pHi attenuated, and an alkaline pHi enhanced, carbachol- and thapsigargin-induced [Ca2+]i influx. We conclude that: (1) an alkaline pHi releases Ca2+ from InsP3-dependent intracellular stores; (2) the store release is InsP3 independent and occurs via an as yet unknown mechanism; (3) the store release stimulates capacitative Ca2+ influx; (4) the capacitative Ca2+ influx activated by InsP3 agonists is decreased by acidic and enhanced by alkaline pHi. The effects of pHi on [Ca2+]i should be of relevance under many physiological conditions.

Attenuation of stimulated Ca2+ influx in colonic epithelial (HT29) cells by cAMP.

In HT29 colonic epithelial cells agonists such as carbachol (CCH) or ATP increase cytosolic Ca2+ activity ([Ca2+]i) in a biphasic manner. The first phase is caused by inositol 1,4,5-trisphophate-(Ins P3-) mediated Ca2+ release from their respective stores and the second plateau phase is mainly due to stimulated transmembraneous Ca2+ influx. The present study was undertaken to examine the effect of increased adenosine 3',5'-cyclic monophosphate (cAMP) (forskolin 10 micromol/l = FOR) on the Ca2+ transient in the presence of CCH (100 micromol/l). In unpaired experiments it was found that FOR induced a depolarization and reduced cytosolic Ca2+ ([Ca2+]i, measured as the fura-2 fluorescence ratio 340/380 nm) significantly. Dideoxyforskolin had no such effect. The effect of FOR was abolished when the cells were depolarized by a high-K+ solution. In further paired experiments utilizing video imaging in conjunction with whole-cell patch-clamp, [Ca2+]i was monitored separately for the patch-clamped cell and three to seven neighbouring cells. In the presence of CCH, FOR reduced [Ca2+]i uniformly from a fluorescence ratio (345/380) of 2.9 +/- 0.12 to 1.8 +/- 0.07 in the patch-clamped cell and its neighbours (n = 48) and depolarized the membrane voltage (Vm) of the patch-clamped cells significantly and reversibly from -54 +/- 7.4 to -27 +/- 5.9 mV (n = 6). In additional experiments Vm was depolarized by 15-54 mV by various increments in the bath K+ concentration. This led to corresponding reductions in [Ca2+]i. Irrespective of the cause of depolarization (high K+ or FOR) there was a significant correlation between the change in Vm and change in [Ca2+]i. These data indicate that the cAMP-mediated attenuation of Ca2+ influx is caused by the depolarization produced by this second messenger.