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J I Vandenberg

Publications and source records attributed to J I Vandenberg.

29 records · Page 2Linked to original sources
PubMed1996

Carbonic anhydrase and cardiac pH regulation.

Membrane-bound carbonic anhydrase (CA) has recently been identified in mammalian cardiac tissue. In this study, we have investigated the histochemical location and functional role of CA in the ferret heart. Heart sections stained by a modified Hansson's technique showed CA to be located on capillary endothelial membranes as well as on sarcolemmal membranes. In the Langendorff-perfused heart, washout of CO2 brought about by switching perfusion between 25 mM HCO3(-)-5% CO2-buffered solution and nominally HCO3(-)-CO2-free solution caused a transient rise in intracellular pH (pHi) measured by the chemical shift of 2-deoxy-D-glucose 6-phosphate with 31P nuclear magnetic resonance spectroscopy. The initial rate of change of pHi, measured over the first 60-75 s of CO2 efflux, was significantly reduced from 0.41 +/- 0.03 pH units/min (n = 9) in control hearts to 0.28 +/- 0.02 pH units/min (n = 5) in the presence of the membrane-permeable CA inhibitor 6-ethoxzolamide (P < 0.05 compared with control) and to 0.22 +/- 0.04 pH units/min (n = 5) in the presence of the membrane-impermeable CA inhibitor CL-11,366 (P < 0.01 compared with control). After reperfusion of the ischemic myocardium, both CA inhibitors caused a significant slowing of initial rate of change in pH (and initial rate of recovery of contractile function) compared with control hearts. These results suggest that CA, by facilitating the hydration-dehydration of CO2-H2CO3, alters the relative concentrations of CO2 inside and outside the cells, thus enhancing the rate of CO2 transfer from the intracellular to extracellular compartments, which contributes significantly to pHi recovery after reperfusion of the ischemic myocardium.

Animals↗
PubMed1995

Extracellular osmotic pressure modulates sodium-calcium exchange in isolated guinea-pig ventricular myocytes.

1. The sensitivity of the cardiac Na(+)-Ca2+ exchange current to changes in osmotic pressure was investigated in guinea-pig ventricular myocytes, using the whole-cell patch-clamp technique. 2. A hyposmotic challenge applied by removal of sucrose from the standard bathing solution reduced exchanger current, measured as the Ni(2+)-sensitive component of whole-cell transsarcolemmal current. These changes were fully reversible. 3. No response of whole-cell current to hyposmosis was observed when Ca2+ was removed from the bathing solution by chelation with 1 mM EGTA. 4. Inclusion of 25 microM exchanger inhibitory peptide (XIP) in the pipette solution caused a marked reduction in the Ni(2+)-sensitive component of membrane current, but the percentage change in Ni(2+)-sensitive membrane slope conductance evoked by hyposmosis was the same as when XIP was omitted from the pipette solution. 5. Exposure of cells to hyperosmotic solutions produced variable responses. In a majority of cells, solutions 30% hyperosmotic compared with control evoked a persistent increase in exchanger current, whereas for solutions 50% hyperosmotic, a larger but transient increase in current was observed. 6. Over a wide range of osmolalities (50-130% of isosmotic) the changes in Ni(2+)-sensitive membrane slope conductance were linearly related to the changes in extracellular osmotic pressure. 7. We propose that one consequence of exposing ventricular myocytes to anisosmotic solutions is modulation of Na(+)-Ca2+ exchange current.

Animals↗
PubMed1995

Cell swelling has differential effects on the rapid and slow components of delayed rectifier potassium current in guinea pig cardiac myocytes.

Cell swelling has been shown to cause activation of a variety of cardiac sarcolemmal ionic conductances including potassium channels. The aim of this study was to investigate the effect of swelling on the two subtypes of delayed rectifier potassium current (IKr and IKs) in single guinea pig myocytes using the whole-cell configuration of the patch clamp technique. When the holding potential was set at -40 mV and stepped to +40 mV for 1 s under isoosmotic conditions (300 mOsm) a delayed rectifier current (IK) was activated (0.86 +/- 0.05 nA; n = 43). Switching to a hypoosmotic solution (200 mOsm) caused a rapid increase in IK to a mean value of 1.43 +/- 0.10 nA (p < 0.05; n = 43). The effect of swelling on the two subtypes of IK was studied by analysis of deactivating tail currents using an envelope of tails protocol (stepping from -40 to +40 mV for 18 different pulse durations between 50 ms and 2.9 s; n = 16). Swelling caused a decrease in current amplitude measured at the end of the pulse (and IKtail) at short durations (< or = 150 ms) however, when the pulse duration was > 1 s swelling caused a significant increase in current. Using a pulse protocol to measure IKr with minimal contamination by IKs (voltage step from -40 to -10 mV for 250 ms) a 50-100 pA current was elicited which could be completely blocked by dofetilide (0.2 microM; n = 3). Introduction of hypoosmotic solution caused a significant decrease in IKr and when dofetilide (0.2 or 1.0 microM) was introduced the current remaining was decreased further (p < 0.05; n = 5), but was not completely blocked, thus suggesting that swelling had decreased the ability of dofetilide to block IKr. Similar results were obtained over a range of dofetilide concentrations and with a second IKr blocker, La3+. In Ca(2+)-free external solutions, pulsing to -10 mV for 500 ms to measure IKr in the absence of IKs, and to +60 mV for 5 s (with 0.2 microM dofetilide) to evoke only IKs, it was clear that swelling significantly increased IKs (pulse and tail currents) and decreased IKr. In addition, when measured using the perforated patch method, swelling modulated IKt and IKs in a similar fashion. We conclude that swelling has differential effects on the subtypes of the classical cardiac IK, which may have important implications in our understanding of the mechanisms underlying ischaemia- and reperfusion-induced arrhythmogenesis.

Animals↗
PubMed1994

Swelling-activated and isoprenaline-activated chloride currents in guinea pig cardiac myocytes have distinct electrophysiology and pharmacology.

We have used the whole-cell patch clamp recording technique to characterize a swelling-activated chloride current in guinea pig atrial and ventricular myocytes and to compare the electrophysiological and pharmacological properties of this current with the isoprenaline-activated chloride current in the same cell types. Osmotic swelling of guinea pig cardiac myocytes caused activation of an outwardly rectifying, anion-selective current with a conductance and permeability sequence of I- approximately NO3- > Br- > Cl- > Asp-. This current was inhibited by tamoxifen, 4,4'-diisothiocyano-stilbene-2,2'-disulphonate and anthracene-9-carboxylic acid, in decreasing order of potency. The isoprenaline-activated anion current, like the swelling-activated current, had a higher permeability to I- relative to Cl-, but it had a markedly reduced conductance for I- compared to Cl-. The isoprenaline-activated current was insensitive to inhibition by tamoxifen, 4,4'-diisothiocyanostilbene-2,2'-disulphonate and anthracene-9-carboxylic acid. The swelling-activated current could be elicited in > 90% atrial myocytes studied but only 34% ventricular myocytes. Conversely, the isoprenaline-activated current was elicited in < 10% atrial myocytes and > 90% ventricular myocytes. In those ventricular myocytes where it was possible to elicit swelling-activated and isoprenaline-activated currents simultaneously, the currents retained the same distinguishing characteristics as when they were elicited in isolation. Thus, while guinea pig atrial cells appear to preferentially express swelling-activated chloride channels and guinea pig ventricular myocytes preferentially express isoprenaline-activated chloride channels, the presence of these two channel types are not necessarily mutually exclusive. This raises the possibility that there may be coordinated regulation of the expression of different Cl- channels within the heart.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1994

Intracellular pH recovery during respiratory acidosis in perfused hearts.

Na(+)-H+ exchange and Na(+)-dependent HCO3- influx both contribute to recovery of intracellular pH (pHi) after an acidosis induced by using the NH4Cl prepulse technique in mammalian and avian cardiac tissue. We have investigated the relative contributions of these mechanisms to pHi recovery during respiratory acidosis in the Langendorff-perfused ferret heart with and without correction of extracellular pH (pHo). pHi was measured from the chemical shift of the exogenous 31P nuclear magnetic resonance pH indicator 2-deoxy-D-glucose 6-phosphate. Intrinsic intracellular buffering capacity, calculated from the change in intracellular HCO3- concentration after a change in CO2, was reduced from approximately 33 (no inhibitors of acid extrusion present) to 19 +/- 5 mM when H+ extrusion during the acid loading phase was inhibited. During respiratory acidosis (pHo approximately 6.95), the proton efflux rate (JH) calculated at pHi 6.85 was 0.30 +/- 0.04 mmol.l-1.min-1 (n = 9). When pHo was corrected by increasing external HCO3- concentration to 60 mM during respiratory acidosis (pHo approximately 7.33), JH was 1.11 +/- 0.11 mmol.l-1.min-1 (n = 7), and when pHo was partially corrected by the addition of 50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid to the perfusion solution (pHo approximately 7.1), JH was 0.64 +/- 0.08 mmol.l-1.min-1 (n = 6). In all three groups Na(+)-H+ exchange and HCO3- influx each contributed approximately 50% to acid-equivalent efflux.(ABSTRACT TRUNCATED AT 250 WORDS)

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1993

Regulation of intracellular pH in the perfused heart by external HCO3- and Na(+)-H+ exchange.

Both Na(+)-dependent HCO3- influx and the Na(+)-H+ antiport have been shown to contribute to recovery from intracellular acidosis in avian and mammalian cardiac tissue. We have investigated the participation of these mechanisms in the recovery of intracellular pH (pHi) after an acid load in the Langendorff-perfused ferret heart. pHi was measured from the phosphorus-31 nuclear magnetic resonance chemical shift of 2-deoxy-D-glucose 6-phosphate. Basal pHi was higher in HCO(3-)-buffered solution (7.05 +/- 0.01; n = 8) than in nominally HCO(3-)-free N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) solution (6.98 +/- 0.02; n = 9). Addition of 5-(N-ethyl-N-isopropyl)amiloride (EIPA) caused a significant fall in pHi in HEPES solution (6.91 +/- 0.02; n = 5) but not in HCO3- solution (7.02 +/- 0.02; n = 5). Intrinsic intracellular buffering capacity in 0 Na(+)-HEPES solution was 37 +/- 2 mmol/l (n = 4), and additional buffering due to HCO(3-)-CO2 was approximately 13 mmol/l in HCO3- solution. After an intracellular acidosis induced by an NH4Cl prepulse, the proton efflux rate (JH) at pHi 6.90 was 0.5 +/- 0.2 nmol.l-1.min-1 (n = 14) in HEPES solution and 1.2 +/- 0.4 mmol.l-1.min-1 (n = 13) in HCO3- solution. The addition of 1 microM EIPA effectively blocked proton efflux in HEPES solution (JH < 0.1 mmol.l-1.min-1; n = 8), whereas it slowed pHi recovery in HCO3- solution (JH = 0.6 +/- 0.2 mmol.l-1.min-1; n = 9). There was no recovery of pHi in Na(+)-free HCO3- solution (JH < 0.1 mmol.l-1.min-1; n = 3). The Na(+)-H+ antiport and a mechanism requiring both external Na+ and HCO3- each contribute approximately 50% to proton efflux at pHi 6.90 during the recovery from intracellular acidosis in the isolated perfused mammalian heart.

Acids↗
PubMed1993

Mechanisms of pHi recovery after global ischemia in the perfused heart.

A Na(+)-HCO3- coinflux carrier and the Na(+)-H+ antiport have both been shown to contribute to recovery from intracellular acidosis in cardiac tissue. We have investigated the participation of these mechanisms as well as metabolite (lactate and CO2) washout in the recovery of pHi after myocardial ischemia. Isovolumic ferret hearts were Langendorff-perfused with either HCO3(-)-buffered or nominally HCO3(-)-free (HEPES-buffered) medium at 30 degrees C. pHi was estimated from the chemical shift of the 31P-nuclear magnetic resonance signal of intracellular PO4-, and net H+ efflux rates were calculated at pHi 6.80. The H+ efflux rate during reperfusion, after 10 minutes of global ischemia, was 15.5 +/- 1.9 mmol.l-1 x min-1 (n = 10) in hearts perfused with HCO3(-)-buffered medium and 8.2 +/- 1.5 mmol.l-1 x min-1 (n = 9, p < 0.01) in hearts perfused with HEPES-buffered medium. HCO3- influx, assessed either by inhibition by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (20 microM) or by initially perfusing hearts with HEPES-buffered medium but reperfusing with HCO3(-)-buffered medium, accounted for 3.5-4.9 mmol.l-1 x min-1, and CO2 efflux accounted for 3.8-6.2 mmol.l-1 x min-1 of the additional H+ efflux in HCO3(-)-buffered medium. H(+)-coupled lactate efflux, measured by NAD(+)-linked spectrophotometric assay and inhibited by the sarcolemmal monocarboxylate transport inhibitor 4,4'-dibenzamidostilbene-2,2'-disulfonate (0.25 mM), contributed 3.7-6.2 mmol.l-1 x min-1. H+ efflux via the 5-(N-ethyl-N-isopropyl)amiloride-sensitive Na(+)-H+ antiport was 1.0-2.9 mmol.l-1 x min-1. pHi recovery after ischemia is therefore principally mediated by metabolite (lactate and CO2) washout. Na(+)-coupled acid extrusion contributed approximately 35% of total H+ efflux in this system. However, the associated Na+ entry (approximately 5 mmol.l-1 x min-1) may contribute to Ca2+ overload after reperfusion.

Animals↗
PubMed1986

Application of progress curve analysis to in situ enzyme kinetics using 1H NMR spectroscopy.

The steady-state kinetics of enzymes in tissues, cells, and concentrated lysates can be characterized using high-resolution nuclear magnetic resonance spectroscopy; this is possible because almost invariably there are differences in the spectra of substrates and products of a reaction and these spectra are obtainable even from optically opaque samples. We used 1H spin-echo NMR spectroscopy to study the hydrolysis of alpha-L-glutamyl-L-alanine by cytosolic peptidases of lysed human erythrocytes. Nonlinear regression of the integrated Michaelis-Menten expression onto the progress-curve data yielded, directly, estimates of Vmax and Km for the hydrolase; a procedure for analyzing progress curves in this manner was adapted and compared with a commonly used procedure which employs the Newton-Raphson algorithm. We also performed a sensitivity analysis of the integrated Michaelis-Menten expression; this yielded equations that indicate under what conditions estimates of Km and Vmax are most sensitive to variations in experimental observables. Specifically, we showed that the most accurate estimates of the steady-state parameters from analysis of progress curves are obtained when the initial substrate concentration is much greater than Km. Furthermore, estimates of these parameters obtained by such an analysis are most sensitive to data obtained when the reaction is 60-80% complete, having started with the highest practicable initial substrate concentration.

Dipeptides↗
PubMed1985

Enkephalin degradation by human erythrocytes and hemolysates studied using 1H NMR spectroscopy.

High resolution (400 MHz) 1H spin-echo NMR spectroscopy was used to monitor the degradation of leucine-enkephalin, and peptide fragments of it, by human erythrocytes and hemolysates. We showed that leucine-enkephalin is rapidly degraded by the cytosolic peptidases of the human erythrocyte, and we have elucidated the most probable pathway of degradation. Computer simulations of the proposed pathway, using a model incorporating the experimentally derived steady-state kinetic parameters obtained for the individual enzyme steps, showed close agreement with the experimental results. From a methodological perspective, the work demonstrates the value of 1H spin-echo NMR spectroscopy for rapidly elucidating, both qualitatively and quantitatively, an entire peptide-degradation pathway as it operates in situ.

Amino Acid Sequence↗
PubMed1985

The assimilation of tri- and tetrapeptides by human erythrocytes.

Evidence is presented that tripeptides enter human erythrocytes via saturable transport system(s) at rates similar to those previously described for dipeptides (King, G.F. and Kuchel, P.W. (1985) Biochem. J. 227, 833-842) but that the transmembrane flux rates for tetrapeptides are considerably less. 1H spin-echo NMR spectroscopy was used to monitor the coupled uptake and hydrolysis of peptides by red cells, since it enabled the simultaneous measurement of the levels of substrates and products of peptidase-catalysed reactions in suspensions with haematocrits similar to those found in vivo. Weighted non-linear least-squares regression of the integrated Michaelis-Menten equation onto progress curves obtained from the hydrolysis of Tyr-Gly-Gly and Gly-Gly-Gly in RBC lysates gave Km = 2.11 +/- 0.08 and 23.4 +/- 0.9 mmol/l and Vmax = 307 +/- 3 and 905 +/- 22 mmol/h per 1 packed cells, respectively. In whole cell suspensions, the rate of hydrolysis was considerably less and was dominated by the transmembrane flux of tripeptide. Progress curve analysis thus yielded the steady-state kinetic parameters for peptide transport; the values were Km = 11.6 +/- 1.1 and 56 +/- 18 mmol/l and Vmax = 12.9 +/- 3.0 and 36.4 +/- 3.2 mmol/h per 1 packed cells, respectively, for the previously mentioned peptides. The rate of transport of the tetrapeptide Gly-Gly-Gly-Gly was considerably less than either of the tripeptides. The above mentioned steady-state kinetic parameters were used in computer simulations of the coupled uptake and hydrolysis of tripeptides by human erythrocytes under physiological conditions; these simulations revealed certain similarities between the rates of peptide uptake by erythrocytes and the intestine in vivo.

Biological Transport, Active↗