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J Gloy

Publications and source records attributed to J Gloy.

9 recordsLinked to original sources
PubMed1998

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↗
PubMed1998

Catecholamines modulate podocyte function.

The aim of this study was to investigate the influence of adrenoceptor agonists on the intracellular calcium activity ([Ca2+]i), membrane voltage (Vm), and ion conductances (Gm) in differentiated mouse podocytes. [Ca2+]i was measured by the Fura-2 fluorescence method in single podocytes. Noradrenaline and the alpha 1-adrenoceptor agonist phenylephrine induced a reversible and concentration-dependent biphasic increase of [Ca2+]i in podocytes (EC50 approximately 0.1 microM for peak and plateau), whereas the alpha 2-adrenoceptor agonist UK 14.304 did not influence [Ca2+]i. The [Ca2+]i response induced by noradrenaline was completely inhibited by the alpha 1-adrenoceptor antagonist prazosin (10 nM). In a solution with a high extracellular K+ (72.5 mM), [Ca2+]i was unchanged and the [Ca2+]i increase induced by noradrenaline was not inhibited by the L-type Ca2+ channel blocker nicardipine (1 microM). Vm and Gm were examined with the patch-clamp technique in the slow whole-cell configuration. Isoproterenol, phenylephrine, and noradrenaline depolarized podocytes and increased Gm. The order of potency for the adrenoceptor agonists was isoproterenol (EC50 approximately 1 nM) > noradrenaline (EC50 approximately 0.3 microM) > phenylephrine (EC50 approximately 0.5 microM). The beta 2-adrenoceptor antagonist ICI 118.551 (5 to 100 nM) inhibited the effect of isoproterenol on Vm. Stimulation of adenylate cyclase by forskolin mimicked the effect of isoproterenol on Vm and Gm (EC50 approximately 40 nM). Isoproterenol induced a time- and concentration-dependent increase of cAMP in podocytes. The effect of isoproterenol was unchanged in the absence of Na+ or in an extracellular solution with a reduced Ca2+ concentration, whereas it was significantly increased in an extracellular solution with a reduced Cl- concentration (from 145 to 32 mM). The data indicate that adrenoceptor agonists regulate podocyte function: They increase [Ca2+]i via an alpha 1-adrenoceptor and induce a depolarization via a beta 2-adrenoceptor. The depolarization is probably due to an opening of a cAMP-dependent Cl- conductance.

Adrenergic alpha-Agonists↗
PubMed1997

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.

Angiotensin II↗
PubMed1997

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.

Adenosine Triphosphate↗
PubMed1996

Swelling of rat mesangial cells induces a Ca2+-dependent Cl- conductance.

Membrane voltage (Vm) and ion currents of rat mesangial cells in primary culture were measured with the patch-clamp technique in the fast whole-cell configuration. Vm was -44 +/- 1 mV (n = 138). A reduction of the osmolality from 290 to 190 mosmol/kg depolarized Vm from -44 +/- 1 to -29 +/- 1 mV (n = 118) and increased the inward and outward conductances (Gm) from 14 +/- 2 to 39 +/- 4 nS and 13 +/- 2 to 37 +/- 4 nS (n = 84), respectively. During the hypotonicity-induced depolarization the cell capacitance increased significantly from 33 +/- 3 to 42 +/- 4 pF (n = 40). The effect of hypotonic cell swelling on Vm was increased in a bath with a reduced extracellular Cl- of 32 mmol/l (by 71 +/- 4%, n = 23), indicating that a Cl- conductance was activated. The permselectivity of this conductance was I- > or = Br- > Cl-. The Vm response was not affected in the presence of a reduced extracellular Na+ of 5 mmol/l (n = 13) and was inhibited in a solution with reduced extracellular Ca2+ concentration (by 63 +/- 9%, n = 14). In microfluorescence measurements with the Ca2+-sensitive dye fura-2 hypotonic cell swelling induced a sustained increase of the intracellular Ca2+ activity, [Ca2+]i (n = 19). The increase of [Ca2+]i was completely inhibited when the extracellular solution was free of Ca2+. The Vm response to hypotonic cell swelling was not attenuated in the presence of the L-type Ca2+ channel blockers nicardipine (n = 5), nifedipine (n = 5) and verapamil (n = 5) (all at 1 micromol/l). The data indicate that in rat mesangial cells, osmotic swelling induces a Ca2+ influx from extracellular space. This Ca2+ influx activates a Cl- conductance resulting in a depolarization of Vm. The enhanced Cl- conductance may lead to KCl extrusion and hence regulatory volume decrease.

Animals↗
PubMed1996

Hydrogen peroxide increases the intracellular calcium activity in rat mesangial cells in primary culture.

Oxygen radicals are known to be mediators of renal injury under several pathophysiological conditions. We have examined the effect of hydrogen peroxide (H2O2) on intracellular calcium activity ([Ca2+]i) in mesangial cells in primary culture. Mesangial cells were loaded with 1 mumol/liter fura-2, and kept in a Ringer-like solution. Fura-2 fluorescence was measured in an inverted microscope at 37 degrees C. Angiotensin II (0.1 nmol/liter) and ATP (0.1 mumol/liter) induced a rapid transient increase of [Ca2+]i, which was followed by a sustained plateau (N = 37 and N = 24). In contrast, the addition of H2O2 (0.01 to 10 mmol/liter, N = 157) caused a time- and concentration-dependent slow increase of [Ca2+]i, which reached a stable [Ca2+]i plateau after 3 to 10 minutes (ED50: 100 mumol/liter). After the removal of H2O2 [Ca2+]i decreased partially and reached a stable value approximately 90% above the resting [Ca2+]i value. Addition of 100 mumol/liter H2O2 to an extracellular Ca(2+)-free solution resulted either in no rise of [Ca2+]i in some experiments (N = 7), or [Ca2+]i oscillations in others (N = 10). In the presence of H2O2 (> 25 mumol/liter), the angiotensin II or ATP mediated increases in [Ca2+]i were almost completely inhibited (N = 15 and N = 10). The cations Ni2+ and La3+ and the Ca(2+)-antagonist verapamil (10 mumol/liter) did not inhibit the H2O2 mediated increase of -Ca2+-i (N = 6 to 9). Flufenamate (100 mumol/liter), an inhibitor of non-selective cation channels inhibited the H2O2 induced increase of [Ca2+]i by 63 +/- 11% (N = 7). Preincubation of the cells with a disulphide reducing agent (dithiothreitol, 500 mumol/liter, N = 5) or an iron-chelator (deferoxamine, 100 mumol/liter, N = 5) attenuated the H2O2 mediated effect by 95 +/- 15% and 74 +/- 6%, respectively. The H2O2 mediated [Ca2+]i increase was completely inhibited when mesangial cells were preincubated with 1 mumol/liter U-83836E, an inhibitor of lipid peroxidation (N = 7), and inhibited by 84 +/- 6% when the cells were pretreated with 1 mmol/liter pyruvate (N = 5). The data indicate that H2O2: (i) increases [Ca2+]i in mesangial cells by a mechanism distinct from angiotensin II or ATP and (ii) that it inhibits the [Ca2+]i response to both agonists.

Adenosine Triphosphate↗
PubMed1993

Effect of extracellular ATP on contraction, cytosolic calcium activity, membrane voltage and ion currents of rat mesangial cells in primary culture.

1. The effects of extracellular ATP on contraction, membrane voltage (Vm), ion currents and intracellular calcium activity [Ca2+]i were studied in rat mesangial cells (MC) in primary culture. 2. Addition of extracellular ATP (10(-5) and 10(-4) M) to MC led to a cell contraction which was independent of extracellular calcium. 3. Membrane voltage (Vm) and ion currents were measured with the nystatin patch clamp technique. ATP induced a concentration-dependent transient depolarization of Vm (ED50: 2 x 10(-6) M). During the transient depolarization ion currents were monitored simultaneously and showed an increase of the inward- and outward current. 4. In a buffer with a reduced extracellular chloride concentration (from 145 to 30 mM) ATP induced a depolarization augmented to -4 +/- 4 mV. 5. ATP-gamma-S and 2-methylthio-ATP depolarized Vm to the same extent as ATP, whereas alpha,beta-methylene-ATP (all 10(-5) M) had no effect on Vm. 6. The Ca2+ ionophore, A23187, depolarized Vm transiently from -51 +/- 2 to -28 +/- 4 mV and caused an increase of the inward current. 7. The intracellular calcium activity [Ca2+]i was measured with the fura-2 technique. ATP stimulated a concentration-dependent increase of [Ca2+]i (ED50: 5 x 10(-6) M). The increase of [Ca2+]i was biphasic with an initial peak followed by a sustained plateau. 8. The [Ca2+]i peak was still present in an extracellular Ca(2+)-free buffer, whereas the plateau was abolished. Verapamil (10(-4) M) did not inhibit the [Ca2+]i increase induced by ATP. 9. The data indicate that extracellular ATP contracts MC and is able to increase [Ca2+]i by the release of Ca2+ from intracellular stores and recruitment from the extracellular space. In addition ATP depolarizes Vm of MC by activating a Cl- conductance. The ATP-induced depolarization is mediated by a P2y receptor.

Adenosine Triphosphate↗
PubMedDate not supplied

Influence of cell culture conditions and passage number on the response of membrane voltage to ATP and angiotensin II in rat mesangial cells.

The influence of passage number and different culture conditions on the effect of ATP and angiotensin II (A II) on membrane voltage (Vm) of rat mesangial cells (MC) was examined with the patch clamp technique in slow and fast whole cell recordings. MC were characterized immunologically and grown in standard medium in primary culture (PC) and long-term culture up to passage 21 in the presence of 90 g/l fetal calf serum (LTC/+FCS) or without or with 5 g/l FCS for 1-3 days (LTC/-FCS). In all three series the studies were performed in a FCS-free Ringer-like solution. Vm of MC did not differ in the series (PC: -49 +/- 1 mV, n = 151; LTC/+FCS: -52 +/- 1 mV, n = 49; LTC/-FCS: -51 +/- 1 mV, n = 44). In primary culture and long-term cultured MC up to passage 8, FCS (ED50 approximately 5 g/l), ATP (ED50 approximately 2 x 10(-6) mol/l) and A II (ED50 approximately 5 x 10(-10) mol/l) induced a depolarization of Vm. Reduction of extracellular Cl- concentration (from 145 to 32 mmol/l) had no effect on Vm but led to an increased depolarization of Vm by FCS, ATP and A II. In long-term cultured MC above passage 8 grown with 90 g/l FCS both ATP and A II induced a concentration-dependent hyperpolarization of Vm, which was attenuated in increased extracellular K+ concentration (from 3.6 to 33.6 mmol/l). In long-term cultured MC beyond passage 8, grown without or with a reduced FCS concentration of 5 g/l, ATP and A II led to a transient depolarization of Vm, which was increased in the presence of 32 mmol/l extracellular Cl-. The depolarization was followed by a hyperpolarization, which was attenuated in the presence of increased extracellular K+. The data indicate that vasoactive agents depolarize Vm of MC in primary culture by activating a Cl- conductance, whereas they hyperpolarize Vm by activation of a K+ conductance in long-term cultured MC grown with FCS. The latter effect was partially reversed when FCS was omitted.

Adenosine Triphosphate↗