Abstract Neuronal uptake1 constitutes the main elimination process of cardiac norepinephrine under normoxic conditions. Uptake1 may be subject to changes during myocardial ischemia. We therefore studied the regulation of the uptake1 carrier in isolated perfused rat hearts, comparing ischemic and nonischemic conditions. Radioligand binding with [3H]mazindol was used to determine carrier densities and affinities, whereas cardiac clearance of [3H]norepinephrine served as a measure of the transport capacity of the uptake1 carrier. When exocytotic norepinephrine release was induced in nonischemic rat hearts by electrical field stimulations, we observed an increase in the cardiac density of uptake1 carriers (Bmax) to 210±5 fmol/mg protein (versus 134±3 fmol/mg in control hearts). Simultaneously, the cardiac clearance of [3H]norepinephrine increased to 41±4% versus 30±4% in control hearts. Both carrier density and norepinephrine clearance returned to baseline values within a period of 40 minutes after stimulation. Carrier affinities (Kd values) did not differ between the groups. Stop-flow ischemia induced a substantial overflow of norepinephrine by itself. Additionally, carrier density was increased to 144% after 40 minutes of stop-flow ischemia (P<.005 versus control hearts). When ischemia was followed by 20 minutes of reperfusion, the Bmax of the uptake1 carrier remained significantly elevated. With a further extension of the reperfusion period to 40 minutes, however, carrier density declined to baseline values. Kd values were not influenced by any of these interventions. Clearance of [3H]norepinephrine was suppressed (to 5±2%) in the first minutes of reperfusion, which may reflect the inverse transport direction of the norepinephrine carrier known to occur in ischemia. After 20 minutes of reperfusion, clearance increased to 39±5% (P<.005 versus control hearts) and then fell to 29±5% after 40 minutes of reperfusion (NS). These results demonstrate that after both electrical field stimulation and myocardial ischemia, the density of uptake1 carrier proteins temporarily increases, which may result in an increased transport capacity for norepinephrine.
Under physiological conditions, norepinephrine is eliminated from the cardiac synaptic cleft primarily via the presynaptic neuronal uptake1 carrier protein, whereas extraneuronal uptake2 is of minor importance for cardiac norepinephrine elimination.1 Uptake1 also plays a pivotal role in disturbances of sympathetic neurotransmission during myocardial ischemia. As previously documented in our laboratory,2 an increased elimination of norepinephrine via the uptake1 carrier is observed during the first minutes of ischemia. Starting 10 minutes after the onset of ischemia, a local metabolic, desipramine-sensitive release of norepinephrine into the synaptic cleft occurs, which is due to a nonexocytotic release of norepinephrine.3 This release is caused by a transport of norepinephrine via the neuronal uptake1 carrier, which reverses its transport direction in consequence of a rapid alteration of the transmembranous sodium gradient during ischemia.4 So far, it is unclear whether ischemia affects not only the function but also the density of uptake1 proteins. Several reports have shown that alterations of synaptic norepinephrine concentrations may change the density of uptake1 carriers, exerting a substantial impact on the transport capacity for norepinephrine.5 6 For postsynaptic α- and β-adrenergic receptors, a significant increase in receptor density has been described during early myocardial ischemia.7 8 It is possible that other membrane proteins involved in sympathoadrenergic signal transduction might also be regulated during cardiac ischemia.
To elucidate the changes in cardiac norepinephrine elimination under ischemic conditions, we studied the uptake1 carrier protein during experimental ischemia in isolated perfused rat hearts. The density and affinity of this protein were measured by radioligand binding. The transport capacity was determined by measuring the clearance of [3H]norepinephrine following ischemia and during reperfusion.
Materials and Methods
Perfused Rat Heart
Male Wistar rats (200 to 250 g) were anesthetized with thiopental (100 mg/kg IP). The thorax was opened and the hearts were rapidly cut out, rinsed in ice-cold buffer, and weighed (mean weight 0.95 g). Thereafter, coronary perfusion was performed according to Langendorff,9 in the absence of external cardiac work at an initial flow rate of 5 mL/min. During each experiment, six hearts were perfused simultaneously by a multichannel peristaltic pump at a constant flow rate. Perfusion was started using a modified Krebs-Henseleit solution (composition in mmol/L: NaCl 125, NaHCO3 16.9, Na2HPO4 0.2, KCl 4.0, CaCl2 1.85, MgCl2 1.0, glucose 11, and EDTA 0.0027). The buffer was gassed with 95% O2 and 5% CO2. pH was adjusted to 7.4 by small variations of the gas flow. The temperature of the perfusion medium was kept at 37.5°C. After an equilibration period of 20 to 30 minutes, ischemia was induced either by stopping perfusion or by limiting flow to 0.5 mL/min. Control preparations were kept at normal flow of 5 mL/min during each experiment. During ischemia, the hearts were covered by a chamber with the temperature regulated to 37.5°C. Low-flow ischemia led to a progressive release of creatine kinase and lactate dehydrogenase from the hearts, whereas normal perfusion did not (not shown, see Reference 1010 ). Reperfusion was induced by returning to a flow of 5 mL/min immediately. For stimulation-evoked release of norepinephrine, the hearts were perfused at a flow rate of 5 mL/min throughout the experiment. Norepinephrine release was induced by electrical field stimulation at 5 V, 6 Hz, and a pulse width of 2 milliseconds from two concave metal paddles of 10×7 mm placed in opposite positions at each heart.10 Hearts continued beating during the experiment. During the process of stimulation, hearts were paced at the respective frequency and regained their spontaneous rhythm thereafter. Six subsequent 1-minute electrical stimulations were performed at intervals of 10 minutes. The exocytotic nature of stimulation-induced norepinephrine release has been previously documented in this model.10 After the experiment, the hearts were immediately frozen in liquid nitrogen. Every intervention was performed with at least six hearts.
Determination of Norepinephrine
Samples from the effluents were taken in 1-minute periods throughout the experiments or within the first 5 minutes of reperfusion after stop-flow ischemia. This time interval has been proved sufficient to recover the norepinephrine overflow during ischemia.3 Norepinephrine was determined in the effluent as previously described.11 Briefly, after a two-step solvent extraction and concentration of the samples, the separation of catecholamines was performed with a reversed-phase high-performance liquid chromatography system. Quantitative analysis was achieved by electrochemical detection. The limit of detection of norepinephrine was 0.1 nmol/L and the recovery was 98%.
Preparation of Cardiac Membranes
For preparation of membranes, whole hearts were cut to pieces with a scalpel, resuspended in ice-cold lysis buffer (5 mmol/L Tris-HCl, pH 7.4, and 2 mmol/L EDTA), and homogenized for 30 seconds in an ultraturrax tissue mincer. The homogenate was centrifuged at 1000g for 15 minutes to remove cell debris and nuclei, and the supernatant was centrifuged twice at 100 000g for 30 minutes. The resulting membrane pellet was resuspended in a buffer containing 50 mmol/L Tris-HCl (pH 7.5), 100 mmol/L NaCl, and 5 mmol/L KCl and used for radioligand binding. Protein concentration was determined according to Bradford.12
Incubation of membranes with [3H]mazindol (NEN-Du Pont) in concentrations ranging from 0.5 to 25 nmol/L was carried out in 50 mmol/L Tris-HCl (pH 7.5), 100 mmol/L NaCl, and 5 mmol/L KCl, with or without 10 μmol/L desipramine to define nonspecific binding, for 20 minutes at 22°C in a volume of 200 μL. On average, 280 μg of protein per tube was used. Mazindol is known to inhibit uptake of dopamine also, but it binds to the dopamine carrier with an affinity ≈10-fold lower than for the uptake1 carrier.13 14 15 In tissues rich in norepinephrine uptake, such as cerebral cortex, binding of [3H]mazindol seems to correspond exclusively to norepinephrine uptake.13 Moreover, comparing the saturation binding of [3H]desipramine to that of [3H]mazindol, we found very similar Bmax values. The nonspecific binding of [3H]desipramine, however, accounted for more than 40% of the total binding; therefore, this radioligand could not be used for our experiments. We also tested the effect of specific dopamine uptake inhibition by GBR-12909, which displays almost 100-fold selectivity for dopamine versus norepinephrine uptake.14 15 In cellular models of cloned, overexpressed carriers,14 15 20 nmol/L GBR-12909 inhibits radioligand binding to dopamine carriers almost completely, whereas it does not affect norepinephrine uptake1.14 15 In cardiac membranes, 20 nmol/L GBR-12909 did not alter [3H]mazindol binding (131±7 fmol/mg protein versus 125±4 fmol/mg in control hearts). However, 100 nmol/L GBR-12909 inhibited binding of [3H]mazindol by 10% (to 110±9 fmol/mg protein), and 500 nmol/L GBR-12909 inhibited binding by 50% (to 65±7 fmol/mg protein). These findings indicate the dependence of cardiac [3H]mazindol binding on norepinephrine uptake1 carriers.
Termination of the Binding Assay
The reaction was terminated by filtration through GF/B filters and washing with ice-cold incubation buffer. Filter radioactivity was determined by liquid scintillation counting. Uptake1 carrier density and affinity were determined from Scatchard plots of the counting data. Fig 1⇓ shows a typical binding experiment. The binding was fully saturable and showed a linear dependence on the amount of membrane protein used. Specific binding depended on the presence of sodium ions and was not detectable in the absence of sodium. Optimum binding was achieved at a concentration of 100 mmol/L NaCl. Specific binding was completely inhibited by saturating concentrations of desipramine, norepinephrine, or nisoxetine, as described in the literature.16 The coefficient of variation between repeated measurements in the same heart was less than 5% in all cases.
Tissues for RNA preparation were taken from ventricular myocardium and the hippocampal brain of three different rats, from three explanted human hearts (ventricular myocardium of patients with dilated cardiomyopathy), and from the peritumorous area of a tissue section removed at the operation of a human brain glioblastoma. RNA was prepared from the frozen tissues according to the protocol of Chomczynski and Sacchi,17 as modified according to our previous publication.18 The amount of RNA in the samples was determined by UV absorption.
Reverse Transcription and PCR
The RNA of each heart was reverse transcribed with mouse multiple lymphoma virus reverse transcriptase (BRL) into cDNA. Sense and antisense oligonucleotide primer pairs were synthesized to match the cDNA sequences of human uptake1 protein14 and the partial sequence of uptake1 protein as cloned from rat pheochromocytoma PC12 cells.19 The primers were chosen to hybridize to both sequences and were therefore matched to highly homologous regions: sense primer, base pairs 230 to 251 of human coding sequence 5′-CAA TGT TTG GCG TTT CCC CTA TC-3′; antisense primer, base pairs 732 to 711 of human coding sequence 5′-CGA CGA CCA TCA GAC AGA GCA-3′. We also amplified the cDNAs for human and rat GAPDH as internal standards, as described.18 Forty cycles of PCR were carried out, and the products were analyzed on an agarose gel. They were then cut out, purified, and sequenced with an automated sequencer.
The measurement of cardiac norepinephrine clearance was carried out as described.20 Briefly, a bolus of [3H]norepinephrine (1 mL, 3 μCi, 100 pmol norepinephrine, NEN-Du Pont) was injected into the perfusion system. Proportional distribution of the radioactivity to the hearts (n=6) and blank channels (n=2) was provided by a mixing chamber and a multichannel peristaltic pump. The effluent was sampled successively at 2-minute intervals before and after the administration of [3H]norepinephrine. Blank channels were additional channels that were perfused freely. Sampling of the radioactivity outflow from these channels was used to control the comparability of the experiments and constituted total [3H]norepinephrine subjected to each heart. A 500-μL portion of each sample was mixed with 4 mL of a liquid scintillation cocktail (Zinsser) and immediately counted in a β-counter. Radioactivity decreased to nearly background levels within 5 minutes after addition of [3H]norepinephrine. The amount of [3H]norepinephrine that was extracted by the hearts (uptake) is expressed as percentage of total [3H]norepinephrine administered.
Results for norepinephrine release are expressed as mean with standard error of the mean (SEM). [3H]Mazindol Bmax data are also given as mean±SEM. For uptake1 carrier affinities, we calculated 95% confidence intervals on the basis of pD2 values. [3H]Norepinephrine extraction data are given as mean with standard deviation (SD). For each experimental condition, six hearts were subjected to a specific intervention, whereas six other hearts were perfused normally for the same time period, without any intervention. Results of time-matched groups were compared by one-way analysis of variance and tested by Scheffé’s test. A value of P<.05 was regarded as significant.
Overflow of Norepinephrine
Release of norepinephrine was measured in the effluent from isolated hearts after various interventions. Samples from the effluents were taken in 1-minute periods or within the first 5 minutes of reperfusion after stop-flow ischemia. Normal perfusion (5 mL/min) was not accompanied by any detectable overflow of norepinephrine (n=24). Electrical field stimulation of the hearts induced a release of norepinephrine, which decreased moderately during subsequent stimulations of the same hearts (n=6). During the first stimulation (S1), we measured a cumulative release of norepinephrine of 101±7 pmol/g in the minutes during and after field stimulation. Subsequent stimulations, at intervals of 10 minutes, led to a norepinephrine overflow of 93±10 (S2), 87±3 (S3), 80±3 (S4), 75±2 (S5), and 67±2 (S6) pmol/g. After stop-flow ischemia (0 mL/min) for 20 minutes, we observed a cumulative release of 245±23 pmol norepinephrine per gram heart weight during the first 5 minutes of reperfusion. Norepinephrine overflow increased to 811±110 pmol/g after 40 minutes of stop-flow ischemia and to 1170±136 pmol/g after 60 minutes of stop-flow ischemia. During 6 hours of low-flow ischemia (0.5 mL/min), the cumulative overflow of norepinephrine was 175±44 pmol/g (0.51±0.12 pmol·min−1·g−1, n=6).
Binding of the Uptake1 Carrier
After stimulation-evoked release of norepinephrine during normal perfusion (six stimulations within 60 minutes), cardiac uptake1 carrier density increased to 165% of control values (P<.001, Fig 2⇓). Within 40 minutes of reperfusion, we observed a normalization of the carrier densities, which had been elevated directly after electrical field stimulation (Fig 2⇓). Carrier affinities (Kd) were not affected under any experimental condition (Table⇓).
After stop-flow ischemia, we detected a gradual increase in the density (Bmax) of uptake1 carrier sites with progression of the ischemic periods (Fig 3⇓). After 80 minutes of stop-flow ischemia, the number of binding sites reached 160% of that of the control hearts and was also elevated after 6 hours of low-flow ischemia (all values, P<.005 versus control values). Addition of 20 nmol/L GBR-12909, a dopamine uptake inhibitor, to binding assays did not affect the upregulation of [3H]mazindol binding sites after 60 minutes of stop-flow ischemia. In the presence of GBR-12909, uptake1 carrier site density was 210±12 fmol/mg in ischemic hearts and 128±10 fmol/mg in normoxic hearts, whereas in the absence of GBR-12909, the density was 190±5 fmol/mg after stop-flow ischemia and 125±4 fmol/mg in control hearts. This finding indicated that norepinephrine uptake1 carriers are specifically upregulated during cardiac ischemia. To study the stability of uptake1 carrier upregulation over time, we induced 40 minutes of stop-flow ischemia and thereafter perfused the hearts at normal flow (Fig 4⇓). It turned out that uptake1 carrier density remained elevated within 20 minutes of reperfusion and returned to baseline values after 40 minutes of reperfusion (Fig 4⇓). Thus, the period of carrier upregulation was comparable to that after electrical field stimulation. The respective carrier affinities (Kd) did not vary significantly between the experimental groups (Table⇑).
The cardiac elimination of norepinephrine was investigated by measuring the clearance of [3H]norepinephrine, which was given as a bolus during reperfusion. In the presence of 10 μmol/L desipramine, virtually no cardiac uptake of [3H]norepinephrine occurred (1.5±1% versus 30±4% in control hearts, Fig 5⇓).
Electrical field stimulation resulted in a significant increase of [3H]norepinephrine clearance (to 41±4%, Fig 5⇑). [3H]Norepinephrine clearance was still elevated 20 minutes after the intervention (40±5%), and it decreased to baseline values 40 minutes after the stimulation (31±7%, Fig 5⇑), paralleling the normalized number of uptake1 carrier sites at that time (Fig 2⇑).
After 40 minutes of stop-flow ischemia, we found a biphasic regulation of cardiac norepinephrine uptake during reperfusion (Fig 6⇓). In the first minutes of reperfusion, cardiac norepinephrine clearance was markedly suppressed (to 5±2% versus 28±3% in control hearts, Fig 6⇓). After 10 minutes of reperfusion, the levels of norepinephrine extraction were still below those of the controls (17±3%, P<.05). After 20 minutes of reperfusion, however, [3H]norepinephrine extraction significantly exceeded that of the control hearts (39±5%, Fig 6⇓), at a time when uptake1 carrier density was still elevated (Fig 4⇑). Norepinephrine clearance reached baseline values after 40 minutes of reperfusion (to 29±5%), paralleling the normalization of the carrier density at that time (Fig 4⇑).
Uptake1 Carrier mRNA
To determine the mRNA expression of the uptake1 carrier, a reverse transcription/polymerase chain reaction was carried out, starting from rat and human heart and brain tissues. The mRNA for GAPDH, a nonregulated housekeeping enzyme, was measured as a control. Fig 7⇓ shows that PCR products in expected sizes were obtained from rat and human brain tissues amplified with the respective primers, which obviously hybridized to both sequences. All PCR products were obtained as single bands that could easily be visualized in ethidium bromide–containing agarose gels. The products from human and rat brains were purified and partially sequenced. They were identical to the expected sequences. In contrast, no specific bands could be amplified from any human or rat heart tissue (n=3 each), although the control message GAPDH was present in all cDNAs (Fig 7⇓).
The present study documents for the first time that the cardiac density of uptake1 carrier proteins undergoes upregulation in membrane preparations from isolated rat hearts subjected to electrical field stimulation or cardiac ischemia. The functional relevance of these findings was demonstrated by a simultaneous increase in the transport capacities for norepinephrine. Both upregulation of the density and increased norepinephrine clearance were reversible within 40 minutes after the interventions.
Until now, it was not known whether changes in uptake1 carrier protein density or affinity during ischemia might contribute to the functional changes of norepinephrine release. The density and affinity of uptake1 carrier proteins can be investigated by studying the binding of tritiated antagonist radioligands to cardiac membranes. Using this assay, we identified a binding site in rat heart membranes to which both desipramine and mazindol bound saturably and with high affinity. Radioligand binding depended on the presence of sodium ions, and it was displaced specifically by uptake1 inhibitors. Previous work has shown that this binding site is directly related to the specific uptake of norepinephrine.13 This assumption was supported by our finding that a specific dopamine-uptake blocker did not affect [3H]mazindol binding to cardiac membranes.
So far, a regulation of the density of carrier proteins has been observed under only a few conditions. Chronic reserpine administration, leading to norepinephrine depletion, reduced the cortical density and transport capacity of uptake1 carrier proteins.5 18 Likewise, treatment with 6-hydroxydopa or clonidine, or surgical denervation, which all reduce norepinephrine release, downregulated uptake1 carrier binding sites in rat brain and heart.6 21 In contrast, chronic monoamine oxidase inhibition5 or administration of amphetamine6 increased the concentration of synaptic norepinephrine and upregulated uptake1 carrier density and transport capacity.
A downregulation of the density of cardiac uptake1 carrier proteins has been described in heart failure,22 23 and this action was accompanied by a decreased norepinephrine uptake, as assessed by in vitro tissue uptake of [3H]norepinephrine22 24 or as measured by the amount of cardiac extraction of norepinephrine.25 A reduced cardiac norepinephrine uptake activity in heart failure has also been documented scintigraphically in vivo.26 It has been assumed that this downregulation may partly explain the elevated synaptic concentrations of norepinephrine in cardiac failure.
We detected a significant upregulation of the density of uptake1 carrier proteins both after cardiac ischemia and after electrical stimulation, resulting in either exocytotic or nonexocytotic norepinephrine release. Our results on the regulation of cardiac uptake1 carrier proteins are in line with observations from other tissues which suggest that elevations of synaptic norepinephrine are accompanied by increased uptake1 carrier densities. For the first time, however, we documented that such a regulation occurred within a very short period of time (40 to 60 minutes) and that the upregulation was fully reversible during reperfusion, both after stop-flow ischemia and after electrical field stimulation.
Corresponding to the pronounced upregulation of uptake1 carrier protein density after electrical field stimulation, we found an increased transport capacity for norepinephrine. Norepinephrine clearance was due to true neuronal uptake1, as indicated by complete blockade of norepinephrine elimination by desipramine. The enhanced transport capacity persisted until 20 minutes after the intervention and declined after 40 minutes, parallel to the normalization of the density of uptake-carrier binding sites. After stop-flow ischemia, the transport capacity for norepinephrine showed fundamentally different characteristics. Initially, cardiac norepinephrine clearance was completely suppressed. In contrast, [3H]norepinephrine extraction significantly exceeded control levels after 20 minutes of reperfusion, when uptake1 carrier density was still increased. Norepinephrine clearance declined to baseline values after 40 minutes, paralleling the normalization of carrier density at that time. The initial suppression of norepinephrine uptake after ischemia can be attributed to the specific disturbance of carrier function in ischemia, because the norepinephrine carrier inverts its net transport direction from inside to outside the neuron, thereby precipitating nonexocytotic norepinephrine release during ischemia. Reversal of the normal transport direction of the carrier is the consequence of a reduced transmembranous sodium gradient in ischemia. Since the sodium gradient slowly recovers during reperfusion,27 it can be concluded that an increased carrier density enables enhanced norepinephrine clearance only in a brief time frame after ischemia.
It was not possible to assess the actual functional consequence of the increased carrier density during ischemia itself because of the inversion of the carrier’s transport direction at that time. It seems plausible, however, that upregulation of the carrier also enhances the outward transport of norepinephrine during ischemia.
The question may be raised which mechanism underlies the upregulation of the norepinephrine carrier in ischemia. Both field stimulation and ischemia induced a significant release of norepinephrine and an upregulation of the cardiac density of uptake1 carrier proteins. Therefore, it is difficult to decide whether this upregulation in ischemia is primarily caused by ischemia itself or whether it is secondary to the increased synaptic concentration of norepinephrine. Generally, the mechanism by which transport proteins are regulated has not yet been clarified. For postsynaptic β-adrenergic receptors, an upregulation during ischemia seems to be accompanied by an increased externalization of receptor proteins.7 The same might happen to carrier proteins during ischemia. It is well known that a rapid loss of energy-rich phosphates occurs in the isolated rat heart during early ischemia.28 29 Consequently, all cellular processes that depend on these phosphates are compromised in ischemia. Cellular mechanisms and enzymes that normally degrade carrier proteins might be hampered, resulting in a higher net surface density of these proteins. A recent publication suggests that the degree of phosphorylation of the norepinephrine transporter regulates its uptake capacity.30 Alternatively, an alteration of ion gradients across the presynaptic membrane might not only influence the transport direction of the carrier but also serve as a stimulus for an enhanced surface expression of carrier proteins.
To examine whether any de novo synthesis of carriers interferes with the observed regulation of their surface density, we also intended to clarify the possible contribution of translation to this process. However, we could not detect any specific mRNA for uptake1 carrier proteins in any of the human or rat hearts investigated. Therefore, we assume that all mRNAs for uptake1 carrier proteins are synthesized and translated in the neural cell soma, which is a considerable distance from the heart, ie, in the stellate ganglion, and that these proteins are then transported to the cardiac nerve ending via axonal transport. Given the short time course of uptake1 carrier protein upregulation during ischemia, it seems highly improbable that an mRNA regulation can be involved in this mechanism.
In conclusion, the overflow of norepinephrine in myocardial ischemia and exocytotic release of norepinephrine are accompanied by a transiently increased density and transport capacity of uptake1 carrier proteins. The mechanism of uptake1 regulation and its consequences for the disturbances of sympathetic neurotransmission in conjunction with and following ischemia remain to be determined.
This work was supported by a grant from the Deutsche Forschungsgemeinschaft (Un 103/1-1). We wish to thank Gertraud Snell and Kai Kronsbein for their excellent technical assistance.
- Received October 23, 1995.
- Accepted February 15, 1996.
- © 1996 American Heart Association, Inc.
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