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PubMed1989

Calcium current is increased in isolated adult myocytes from hypertrophied rat myocardium.

To study the effects of myocardial hypertrophy resulting from chronic pressure overload on excitation-contraction coupling, the cardiac transmembrane L-type calcium current (ICa) was investigated in the Goldblatt renovascular hypertensive (HBP) rat. ICa was measured in single myocytes enzymatically isolated from control (CTRL) and HBP rat hearts using the whole-cell, patch-clamp method. The peak ICa and ICa density (obtained by normalizing ICa to the average cell capacitative surface area) were larger in HBP cells (n = 15) than in CTRL cells (n = 10) at membrane potentials of -20 to 50 mV (p less than 0.01). The maximal peak ICa increased from 0.9 +/- 0.5 nA (mean +/- SD) in CTRL cells to 2.8 +/- 1.0 nA in HBP cells (p less than 0.001). The corresponding ICa density increased from 5.3 +/- 2.7 to 16.2 +/- 6.0 microA/cm2 (p less than 0.001). There was no shift in the current-voltage relation between CTRL and HBP cells. The time course of decay of HBP ICa in response to clamp steps to the plateau range of the action potential (membrane potential, Vm = -10 to 30 mV) was delayed when compared with that of CTRL ICa. The inactivation time constants (biexponential) for the maximal ICa were 6.9 +/- 1.9 and 36.0 +/- 9.3 msec for CTRL cells and 6.7 +/- 1.4 and 49.5 +/- 12.9 msec for HBP cells (p less than 0.05 for the slower component of the maximal ICa). There was no difference in the steady-state inactivation of ICa (f infinity) for the CTRL and HBP cells. From the maximal peak ICa, cytoplasmic free Ca2+ was estimated to reach a pCa of 6.95 +/- 0.07 for CTRL cells and 6.64 +/- 0.13 for HBP cells. It is concluded that ICa is increased with myocardial hypertrophy. The lengthening of the action potential in hypertrophied rat myocardium is due to an increase in peak current density and to the slower inactivation of the maximal ICa. The increased transmembrane flux of Ca2+ via ICa in HBP cells is inadequate to achieve a myoplasmic free Ca2+ level sufficient for direct partial activation of the contractile myofilaments. However, in the scheme of the calcium-triggered calcium release hypothesis such an increase could provide an increased amount of activator calcium and/or serve to amplify the release of Ca2+ from sarcoplasmic reticulum, thereby contributing to preserved peak developed tension in hypertrophied rat myocardium.

Action Potentials↗

PubMed1999

Mechanism of allosteric modulation of rod cyclic nucleotide-gated channels.

The cyclic nucleotide-gated (CNG) channel of retinal rod photoreceptor cells is an allosteric protein whose activation is coupled to a conformational change in the ligand-binding site. The bovine rod CNG channel can be activated by a number of different agonists, including cGMP, cIMP, and cAMP. These agonists span three orders of magnitude in their equilibrium constants for the allosteric transition. We recorded single-channel currents at saturating cyclic nucleotide concentrations from the bovine rod CNG channel expressed in Xenopus oocytes as homomultimers of alpha subunits. The median open probability was 0.93 for cGMP, 0.47 for cIMP, and 0.01 for cAMP. The channels opened to a single conductance level of 26-30 pS at +80 mV. Using signal processing methods based on hidden Markov models, we determined that two closed and one open states are required to explain the gating at saturating ligand concentrations. We determined the maximum likelihood rate constants for two gating schemes containing two closed (denoted C) and one open (denoted O) states. For the C left and right arrow C left and right arrow O scheme, all rate constants were dependent on cyclic nucleotide. For the C left and right arrow O left and right arrow C scheme, the rate constants for only one of the transitions were cyclic nucleotide dependent. The opening rate constant was fastest for cGMP, intermediate for cIMP, and slowest for cAMP, while the closing rate constant was fastest for cAMP, intermediate for cIMP, and slowest for cGMP. We propose that interactions between the purine ring of the cyclic nucleotide and the binding domain are partially formed at the time of the transition state for the allosteric transition and serve to reduce the transition state energy and stabilize the activated conformation of the channel. When 1 microM Ni2+ was applied in addition to cyclic nucleotide, the open time increased markedly, and the closed time decreased slightly. The interactions between H420 and Ni2+ occur primarily after the transition state for the allosteric transition.

Algorithms↗

PubMed2003

Decoding of synaptic voltage waveforms by specific classes of recombinant high-threshold Ca(2+) channels.

Studies suggest that the preferential role of L-type voltage-sensitive Ca(2+) channels (VSCCs) in coupling strong synaptic stimulation to transcription is due to their selective activation of local chemical events. However, it is possible that selective activation of the L-type channel by specific voltage waveforms also makes a contribution. To address this issue we have examined the response of specific Ca(2+) channel types to simulated complex voltage waveforms resembling those encountered during synaptic plasticity (gamma and theta firing frequency). L-, P/Q- and N-type VSCCs (alpha1C, alpha1A, alpha1B/beta1B/alpha2delta, respectively) were all similarly activated by brief action potential (AP) waveforms or sustained step depolarization. When complex waveforms containing large excitatory postsynaptic potentials (EPSPs), APs and spike accommodation were applied under voltage clamp we found that the integrated L-type VSCC current was approximately three times larger than that produced by the P/Q- or N-type Ca(2+) channels (gamma frequency 1 s stimulation). For P/Q- or N-type channels the complex waveforms led to a smaller current than that expected from the response to a simple 1 s step depolarization to 0 or +20 mV. EPSPs present in the waveforms favoured the inactivation of P/Q- and N-type channels. In contrast, activation of the L-type channel was dependent on both EPSP- and AP-mediated depolarization. Expression of P/Q-type channels with reduced voltage-dependent inactivation (alpha1A/beta2A/alpha2delta) or the use of hyperpolarized intervals between AP stimuli greatly increased their response to complex voltage stimuli. We propose that in response to complex synaptic voltage waveforms P/Q- and N-type channels can undergo selective voltage-dependent inactivation leading to a Ca(2+) current mediated predominantly by L-type channels.

Action Potentials↗

PubMed1999

Connexin diversity and gap junction regulation by pHi.

The molecular mechanisms controlling pH-sensitivity of gap junctions formed of two different connexins are yet to be determined. We used a proton-sensitive fluorophore and electrophysiological techniques to correlate changes in intracellular pH (pHi) with electrical coupling between connexin-expressing Xenopus oocytes. The pH sensitivities of alpha 3 (connexin46), alpha 2 (connexin38), and alpha 1 (connexin43) were studied when these proteins were expressed as: 1) nonjunctional hemichannels (for alpha 3 and alpha 2), 2) homotypic gap junctions, and 3) heterotypic gap junctions. We found that alpha 3 hemichannels are sensitive to changes in pHi within a physiological range (pKa = 7.13 +/- 0.03; Hill coefficient = 3.25 +/- 1.73; n = 8; mean +/- SEM); an even more alkaline pKa was obtained for alpha 2 hemichannels (pKa = 7.50 +/- 0.03; Hill coefficient = 3.22 +/- 0.66; n = 13). The pH sensitivity curves of alpha 2 and alpha 3 homotypic junctions were indistinguishable from those recorded from hemichannels of the same connexin. Based on a comparison of pKa values, both alpha 3 and alpha 2 gap junctions were more pHi-dependent than alpha 1. The pH sensitivity of alpha 2-containing heterotypic junctions could not be predicted from the behavior of the two connexons in the pair. When alpha 2 was paired with alpha 3, the pH sensitivity curve was similar to that obtained from alpha 2 homotypic pairs. Yet, pairing alpha 2 with alpha 1 shifted the curve similar to homotypic alpha 1 channels. Pairing alpha 2 with a less pH sensitive mutant of alpha 1 (M257) yielded the same curve as when alpha 1 was used. However, the pH sensitivity curve of alpha 3/alpha 1 channels was similar to alpha 3/alpha 3, while alpha 3/M257 was indistinguishable from alpha 3/alpha 1. Our results could not be consistently predicted by a probabilistic model of two independent gates in series. The data show that dissimilarities in the pH regulation of gap junctions are due to differences in the primary sequence of connexins. Moreover, we found that pH regulation is an intrinsic property of the hemichannels, but pH sensitivity is modified by the interactions between connexons. These interactions should provide a higher level of functional diversity to gap junctions that are formed by more than one connexin.

Animals↗

PubMed2005

The conserved histidine 295 does not contribute to proton cotransport by the glutamate transporter EAAC1.

Transmembrane glutamate transport by the excitatory amino acid carrier (EAAC1) is coupled to the cotransport of three Na(+) ions and one proton. Previously, we suggested that the mechanism of H(+) cotransport involves protonation of the conserved glutamate residue E373. However, it was also speculated that the cotransported proton is shared in a H(+)-binding network, possibly involving the conserved histidine 295 in the sixth transmembrane domain of EAAC1. Here, we used site-directed mutagenesis together with pre-steady-state electrophysiological analysis of the mutant transporters to test the protonation state of H295 and to determine its involvement in proton transport by EAAC1. Our results show that replacement of H295 with glutamine, an amino acid residue that cannot be protonated, generates a fully functional transporter with transport kinetics that are close to those of the wild-type EAAC1. In contrast, replacement with lysine results in a transporter in which substrate binding and translocation are dramatically inhibited. Furthermore, it is demonstrated that the effect of the histidine 295 to lysine mutation on the glutamate affinity is caused by its positive charge, since wild-type-like affinity can be restored by changing the extracellular pH to 10.0, thus partially deprotonating H295K. Together, these results suggest that histidine 295 is not protonated in EAAC1 at physiological pH and, thus, does not contribute to H(+) cotransport. This conclusion is supported by data from H295C-E373C double mutant transporters which demonstrate that these residues cannot be linked by oxidation, indicating that H295 and E373 are not close in space and do not form a proton binding network. A kinetic scheme is used to quantify the results, which includes binding of the cotransported proton to E373 and binding of a modulatory, nontransported proton to the amino acid side chain in position 295.

Amino Acid Substitution↗

PubMed1996

Determination of the solution structure of the SH3 domain of human p56 Lck tyrosine kinase.

The solution structure of the SH3 domain of human p56 Lck tyrosine kinase (Lck-SH3) has been determined by multidimensional heteronuclear NMR spectroscopy. The structure was calculated from a total of 935 experimental restraints comprising 785 distance restraints derived from 1017 assigned NOE cross peaks and 150 dihedral angel restraints derived from 160 vicinal coupling constants. A novel combination of the constant-time 1H-13C NMR correlation experiment recorded with various delays of the constant-time refocusing delays and a fractionally 13C-labelled sample was exploited for the stereospecific assignment of prochiral methyl groups. Additionally, 28 restraints of 14 identified hydrogen bonds were included. A family of 25 conformers was selected to characterize the solution structure. The average root-mean-square deviations of the backbone atoms (N, C alpha, C', O) among the 25 conformers is 0.42 A for residues 7 to 63. The N- and C-terminal residues, 1 to 6 and 64 to 81, are disordered, while the well-converged residues 7 to 63 correspond to the conserved sequences of other SH3 domains. The topology of the SH3 structure comprises five anti-parallel beta-strands arranged to form two perpendicular beta-sheets, which are concave and twisted in the middle part. The overall secondary structure and the backbone conformation of the core beta-strands are almost identical to the X-ray structure of the fragment containing the SH2-SH3 domains of p56 Lck [Eck et al. (1994) Nature, 368, 764-769]. The X-ray structure of the SH3 domain in the tandem SH2-SH3 fragment is spatially included within the ensemble of the 25 NMR conformers, except for the segment of residues 14 to 18, which makes intermolecular contacts with an adjacent SH2 molecule and the phosphopeptide ligand in the crystal lattice. Local structural differences from other known SH3 domains are also observed, the most prominent of which is the absence in Lck-SH3 of the two additional short beta-strands in the regions Ser15 to Glu17 and Gly25 to Glu27 flanking the so-called "RT-Src' loop. This loop (residues Glu17 to Leu24), together with the "n-Src' loop (residues Gln37 to Ser46) confines the ligand interaction site which is formed by a shallow patch of hydrophobic amino acids (His14, Tyr16, Trp41, Phe54 and Phe59). Both loops are flexible and belong to the most mobile regions of the protein, which is assessed by the heteronuclear 15N, 1H-NOE values characterizing the degree of internal backbone motions. The aromatic residues of the ligand binding site are arranged such that they form three pockets for interactions with the polyproline ligand.

Amino Acids↗

PubMed2004

Functional adult myocardium in the absence of Na+-Ca2+ exchange: cardiac-specific knockout of NCX1.

The excitation-contraction coupling cycle in cardiac muscle is initiated by an influx of Ca2+ through voltage-dependent Ca2+ channels. Ca2+ influx induces a release of Ca2+ from the sarcoplasmic reticulum and myocyte contraction. To maintain Ca2+ homeostasis, Ca2+ entry is balanced by efflux mediated by the sarcolemmal Na+-Ca2+ exchanger. In the absence of Na+-Ca2+ exchange, it would be expected that cardiac myocytes would overload with Ca2+. Using Cre/loxP technology, we generated mice with a cardiac-specific knockout of the Na+-Ca2+ exchanger, NCX1. The exchanger is completely ablated in 80% to 90% of the cardiomyocytes as determined by immunoblot, immunofluorescence, and exchange function. Surprisingly, the NCX1 knockout mice live to adulthood with only modestly reduced cardiac function as assessed by echocardiography. At 7.5 weeks of age, measures of contractility are decreased by 20% to 30%. We detect no adaptation of the myocardium to the absence of the Na+-Ca2+ exchanger as measured by both immunoblots and microarray analysis. Ca2+ transients of isolated myocytes from knockout mice display normal magnitudes and relaxation kinetics and normal responses to isoproterenol. Under voltage clamp conditions, the current through L-type Ca2+ channels is reduced by 50%, although the number of channels is unchanged. An abbreviated action potential may further reduce Ca2+ influx. Rather than upregulate other Ca2+ efflux mechanisms, the myocardium appears to functionally adapt to the absence of the Na+-Ca2+ exchanger by limiting Ca2+ influx. The magnitude of Ca2+ transients appears to be maintained by an increased gain of sarcoplasmic reticular Ca2+ release. The myocardium of the NCX1 knockout mice undergoes a remarkable adaptation to maintain near normal cardiac function.

Action Potentials↗

PubMed1996

Vascular graft thrombosis after pancreatic transplantation: univariate and multivariate operative and nonoperative risk factor analysis.

BACKGROUND: Vascular thrombosis is still the leading cause of nonimmunologic, technical pancreatic transplant graft failures. The paucity of published data--coupled with our large institutional experience with pancreatic transplantation in all recipient and transplant categories, using a wide spectrum of surgical techniques--provided the impetus for a retrospective study of graft thrombosis risk factors. STUDY DESIGN: Four hundred thirty-eight patients with pancreatic transplants (45 percent simultaneous pancreas-kidney [SPK], 23 percent pancreas-after-kidney [PAK], and 32 percent pancreatic transplants alone [PTA] and with Type I insulin-dependent diabetes mellitus were studied retrospectively. Of 438 pancreatic transplants, 90 percent were bladder-drained and 10 percent were enteric-drained. Ninety-three percent were from cadaver donors, 90 percent were whole-organ grafts, and 20 percent were retransplantations. Quadruple immunosuppression was given. For bladder-drained, whole-organ transplantations (n=378), we performed Cox regression analyses to study the impact on pancreatic graft thrombosis of donor, recipient, mode of preservation, and surgical variables. RESULTS: The overall thrombosis rate was 12 percent (5 percent arterial, 7 percent venous). For cadaver, bladder-drained, whole-organ pancreatic transplants, the thrombosis incidence was highest in PAK recipients (20 percent). The PAK category was also found to be an independent risk factor for thrombosis by stepwise Cox regression analysis. In separate stepwise Cox regression analyses for each category, other identified risk factors for thrombosis included the following: donor age (PAK, PTA); cardiocerebrovascular cause of donor death (SPK, PAK); the use of an aortic Carrel patch (SPK); arterial pancreatic graft reconstruction using a splenic artery to superior mesenteric artery anastomosis (SPK, PTA) or an interposition graft between the splenic artery and the superior mesenteric artery (PTA); portal vein extension graft (PAK); left-sided implantation into the recipient (PAK); and graft pancreatitis (defined as hyperamylasemia exceeding five days post-transplant [PAK, PTA]). CONCLUSIONS: Older donors or those who died of cardiocerebrovascular disease should not be considered for any recipient category. Preservation time needs to be minimized and strategies need to be developed to decrease graft pancreatitis. Surgically, left-sided implantation and arterial reconstructions other than the Y-graft also increase risk, as do portal vein extensions. Renal transplants alone in prospective PAK recipients with Type I diabetes mellitus should, therefore, always be implanted left-sided to allow for right-sided pancreatic graft placement.

Adult↗

PubMed2000

Cocaine and kindling alter the sensitivity of group II and III metabotropic glutamate receptors in the central amygdala.

G-protein-coupled metabotropic glutamate receptors (mGluRs) are being implicated in various forms of neuroplasticity and CNS disorders. This study examined whether the sensitivities of mGluR agonists are modulated in a distinct fashion in different models of synaptic plasticity, specifically, kindling and chronic cocaine treatment. The influence of kindling and chronic cocaine exposure in vivo was examined in vitro on the modulation of synaptic transmission by group II and III metabotropic glutamate receptors using whole cell voltage-clamp recordings of central amygdala (CeA) neurons. Synaptic transmission was evoked by electrical stimulation of the basolateral amygdala (BLA) and ventral amygdaloid pathway (VAP) afferents in brain slices from control rats and from rats treated with cocaine or exposed to three to five stage-five kindled seizures. This study shows that after chemical stimulation with chronic cocaine exposure or after electrical stimulation with kindling the receptor sensitivities for mGluR agonists are altered in opposite ways. In slices from control rats, group II agonists, (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine (LCCG1) and (+)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY354740), depressed neurotransmission more potently at the BLA-CeA than at the VAP-CeA synapse while group III agonist, L(+)-2-amino-4-phosphonobutyrate (LAP4), depressed neurotransmission more potently at the VAP-CeA synapse than at the BLA-CeA. These agonist actions were not seen (were absent) in amygdala neurons from chronic cocaine-treated animals. In contrast, after kindling, concentration response relationships for LCCG1 and LAP4 were shifted to the left, suggesting that sensitivity to these agonists is increased. Except at high concentrations, LCCG1, LY354740, and LAP4 neither induced membrane currents nor changed current-voltage relationships. Loss of mGluR inhibition with chronic cocaine treatment may contribute to counter-adaptive changes including anxiety and depression in cocaine withdrawal. Drugs that restore the inhibitory effects of group II and III mGluRs may be novel tools in the treatment of cocaine dependence. The enhanced sensitivity to group II and III mGluR agonists in kindling is similar to that recorded at the lateral to BLA synapse in the amygdala where they reduce epileptiform bursting. These findings suggest that drugs modifying mGluRs may prove useful in the treatment of cocaine withdrawal or epilepsy.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed2006

Identification of a disulfide bridge linking the fourth and the seventh extracellular loops of the Na+/glucose cotransporter.

The Na+/glucose cotransporter (SGLT1) is an archetype for the SLC5 family, which is comprised of Na+-coupled transporters for sugars, myo-inositol, choline, and organic anions. Application of the reducing agent dithriothreitol (DTT, 10 mM) to oocytes expressing human SGLT1 affects the protein's presteady-state currents. Integration of these currents at different membrane potentials (Vm) produces a Q-V curve, whose form was shifted by +25 mV due to DTT. The role of the 15 endogenous cysteine residues was investigated by expressing SGLT1 constructs, each bearing a single mutation for an individual cysteine, in Xenopus oocytes, using two-microelectrode voltage-clamp electrophysiology and fluorescent labeling. 12 of the 15 mutants were functional and could be separated into three distinct groups based on the effect of the mutation on the Q-V curve: four mutants did not perturb the transferred charge, six mutants shifted the Q-V curve towards negative potentials, and two mutants (C255A and C511A) produced a shift in the positive direction that was identical to the shift produced by DTT on the wild-type (wt) SGLT1. The double mutant C(255,511)A confirms that the effects of each single mutant on the Q-V curve were not additive. With respect to wt SGLT1, the apparent affinities for alpha-methylglucose (alphaMG) were increased in a similar manner for the single mutants C255A and C511A, the double mutant C(255,511)A as well as for wt SGLT1 treated with DTT. When exposed to a maleimide-based fluorescent probe, wt SGLT1 was not significantly labeled but mutants C255A and C511A could be clearly labeled, indicating an accessible cysteine residue. These residues are presumed to be C511 and C255, respectively, as the double mutant C(255,511)A could not be labeled. These results strongly support the hypothesis that C255 and C511 form a disulfide bridge in human SGLT1 and that this disulfide bridge is involved in the conformational change of the free carrier.

Animals↗

PubMed1998

Direct measurement of SR release flux by tracking 'Ca2+ spikes' in rat cardiac myocytes.

1. Ca2+ release flux across the sarcoplasmic reticulum (SR) during cardiac excitation-contraction coupling was investigated using a novel fluorescence method. Under whole-cell voltage-clamp conditions, rat ventricular myocytes were dialysed with a high concentration of EGTA (4.0 mM, 150 nM free Ca2+), to minimize the residence time of released Ca2+ in the cytoplasm, and a low-affinity, fast Ca2+ indicator, Oregon Green 488 BAPTA-5N (OG-5N; 1.0 mM, Kd approximately 31 microM), to optimize the detection of localized high [Ca2+] in release site microdomains. Confocal microscopy was employed to resolve intracellular [Ca2+] at high spatial and temporal resolution. 2. Analytical and numerical analyses indicated that, under conditions of high EGTA concentration, the free [Ca2+] change is the sum of two terms: one major term proportional to the SR release flux/Ca2+ influx, and the other reflecting the running integral of the released Ca2+. 3. Indeed, the OG-5N transients in EGTA-containing cells consisted of a prominent spike followed by a small pedestal. The OG-5N spike closely resembled the first derivative (d[Ca2+]/dt) of the conventional Ca2+ transient (with no EGTA), and mimicked the model-derived SR Ca2+ release function reported previously. In SR Ca2+-depleted cells, the OG-5N transient also closely followed the waveform of L-type Ca2+ current (ICa). Using ICa as a known source of Ca2+ influx, SR flux can be calibrated in vivo by a linear extrapolation of the ICa-elicited OG-5N signal. 4. The OG-5N image signal was localized to discrete release sites at the Z-line level of sarcomeres, indicating that the local OG-5N spike arises from 'Ca2+ spikes' at transverse (T) tubule-SR junctions (due to the imbalance between calcium ions entering the cytosol and the buffer molecules). 5. Both peak SR release flux and total amount of released Ca2+ exhibited a bell-shaped voltage dependence. The temporal pattern of SR release also varied with membrane voltage: Ca2+ release was most synchronized and produced maximal peak release flux (4.2 mM s-1) at 0 mV; in contrast, maximal total release occurred at -20 mV (71 versus 61 microM at 0 mV), but the localized release signals were partially asynchronous. Since the maximal conventional [Ca2+] transient and contraction were elicited at 0 mV, it appears that not only the amount of Ca2+ released, but also the synchronization among release sites affects the whole-cell Ca2+ transient and the Ca2+-myofilament interaction.

Algorithms↗

PubMed2002

Role of the cardiac Na(+)/H(+)exchanger in [Ca(2+)](i)and [Na(+)](i)handling during intracellular acidosis. Effect of cariporide (Hoe 642).

Intracellular acidosis is one of the alterations occurring in cardiac ischemia and has been discussed to be important in altering excitation--contraction coupling. The aim of this study was to determine how intracellular acidosis may affect intracellular sodium and calcium handling. Cardiomyocytes were isolated from the hearts of adult male guinea-pigs by standard techniques and superfused with modified Tyrode's solution at room temperature, either HEPES buffered containing 10 mM NaHCO(3)or HEPES buffered without NaHCO(3), in order to examine a possible interaction with the sodium bicarbonate symport. The whole cell voltage clamp technique was used utilizing 3 M Omega pipettes filled with (mM): Cs aspartate 120, CsCl 20, MgCl(2)1, NaCl 5, Mg-ATP 2, HEPES 10 and either 100 microM Fura-2 or 100 microM SBFI. The pH of the pipette solution was either 7.2 or 6.5. Cells were kept at a holding potential of -80 mV and after a pre-pulse to -40 mV the membrane was continuously clamped to potentials from -30 to +80 mV in 10 mV steps. Intracellular Ca(2+)or Na(+)were estimated using the Fura-2 or SBFI technique (impermeable salt), respectively. The cardiac Na(+)/H(+)exchanger was inhibited using the Na(+)/H(+)- exchange inhibitor cariporide (Hoe 642) (1 microM), when indicated. In NaHCO(3)-free experiments we found an increase in intracellular sodium reflected by a rise in the SBFI ratio of 0.326 +/- 0.01 upon intracellular acidification, in contrast to cells perfused at pH = 7.2 (no significant increase in intracellular Na(+)) (P< 0.05). There was no difference in intracellular calcium handling between cells perfused with solutions of pH = 7.2 or 6.5 (Fura-2 Delta ratio: 0.79 +/- 0.10 vs 0.82 +/- 0.07, n.s.). The l -type calcium current also remained unchanged. Blockade of the Na(+)/H(+)exchanger by Hoe 642 had no influence on cells perfused at pH = 7.2 but inhibited the increase in intracellular Na(+)at pH = 6.5 (0.023 +/- 0.026 in the presence of Hoe 642 vs 0.326 +/- 0.01 without Hoe 642, P< 0.05) without affecting [Ca(2+)](i)or the L-type calcium current. In cells superfused with a Tyrode solution containing NaHCO(3), the increase in intracellular sodium concentration was even more pronounced. Under these conditions Hoe 642 also antagonized this increase in intracellular sodium but without reaching the control level. We conclude that under these experimental conditions intracellular acidification causes an increase in [Na(+)](i)without changing intracellular Ca(2+)or the L-type calcium current. In addition in bicarbonate-buffered systems the acidosis-induced increase in sodium is enhanced which may involve the Na(+)/HCO(3)(minus sign)symport. The effect of cariporide (Hoe 642) in intracellular acidosis seems to be based on antagonization of the rise in intracellular sodium rather than calcium in this model.

Animals↗