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(Circulation Research. 1996;78:102-109.)
© 1996 American Heart Association, Inc.

Articles

Synergistic Actions of Glucagon and Miniglucagon on Ca²⁺ Mobilization in Cardiac Cells

Anne Sauvadet, Troy Rohn, Françoise Pecker, Catherine Pavoine

From INSERM Unité 99, Hôpital Henri Mondor, Créteil, France.

Correspondence to Dr Catherine Pavoine, INSERM Unité 99, Hôpital Henri Mondor, 94010 Créteil, France.

	Abstract

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Abstract It has been recently shown that the physiological processing of glucagon into its C-terminal (19-29) fragment, miniglucagon, by cardiac cells was essential for the contractile positive inotropic effect of the hormone. However, the mechanisms underlying the effects of miniglucagon remained undetermined. In the present study, we assessed the effects of miniglucagon on Ca²⁺ homeostasis in embryonic chick ventricular myocytes. In quiescent cells, short-term applications of 0.1 nmol/L miniglucagon markedly increased the accumulation of ⁴⁵Ca into intracellular compartments resistant to digitonin lysis and sensitive to caffeine. Ca²⁺ accumulation into the sarcoplasmic reticular (SR) store was further attested by fura 2 imaging studies on quiescent or prestimulated cells: miniglucagon potentiated Ca²⁺ release from the SR compartment triggered by caffeine and evoked a rise in cytosolic Ca²⁺ when applied on cells pretreated with 1 µmol/L thapsigargin, a specific inhibitor of the SR Ca²⁺ pump. Glucagon alone produced a small cytosolic Ca²⁺ signal that was considerably amplified by miniglucagon. The action of glucagon was mimicked by 8-bromo-cAMP and was blocked by isradipine, suggesting that it relied on the activation of L-type Ca²⁺ channels, via phosphorylation. We conclude that the combined actions of miniglucagon and glucagon on Ca²⁺ accumulation into SR stores and Ca²⁺ release from the same stores are likely to support the positive inotropic effect elicited in vivo by glucagon on heart contraction.

Key Words: glucagon • miniglucagon • cardiac cells • Ca²⁺

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
It is known that intravenous administration of glucagon, a 29–amino acid peptide derived from the pancreatic posttranslational processing of proglucagon, exerts a potent positive inotropic action on heart contraction.¹ Accordingly, glucagon is prescribed in cardiac emergency in the face of ß-adrenergic blockade,^{2 3} Ca²⁺ channel blocker–induced myocardial dysfunction,⁴ or tricyclic antidepressant poisoning.⁵

In a previous study, we have suggested that the positive cardiac inotropic effect of glucagon was not only due to the action of the glucagon peptide (1-29) but also relied on that of its C-terminal (19-29) fragment, referred to as miniglucagon.⁶ Miniglucagon is not a circulating peptide. It is generated in the extracellular medium upon incubation of heart or liver cells with glucagon.^{6 7} In the heart cell environment, the accumulation of miniglucagon can reach 6% of the initial glucagon concentration after 8 minutes.⁶ The endopeptidase responsible for the cleavage of the dibasic doublet Arg¹⁷-Arg¹⁸ in the glucagon molecule has been identified as a membranous enzyme sensitive to thiol reagents⁷ and has been purified from the hepatic tissue.⁸

We have shown that glucagon alone, under minimal degradation conditions, has no effect on the contraction of beating chick embryo ventricular cells. In contrast, a 45% increase in the amplitude of cell contractility was seen with the combination of nanomolar concentrations of glucagon and miniglucagon. Interestingly, 8-bromo-cAMP could substitute for glucagon.⁶

These observations implied a dual mechanism for glucagon action: one component related to the action of the glucagon peptide (1-29), involving the activation of the cAMP pathway, through either adenylyl cyclase activation or phosphodiesterase inhibition,^{9 10 11} and another component linked to the action of the metabolite (19-29). The latter did not rely on either cAMP or cGMP⁶ and remained to be defined.

Since Ca²⁺ plays a primary role in cardiac contraction and because we had previously shown that in liver, miniglucagon exerts a biphasic regulation on the plasma membrane Ca²⁺ pump ensuring Ca²⁺ extrusion out of the cell,^{12 13 14} we decided to look for a possible effect of miniglucagon on Ca²⁺ metabolism in heart cells. We show that miniglucagon produces a time-dependent Ca²⁺ accumulation into caffeine-sensitive SR compartment(s) and considerably amplifies the cytosolic Ca²⁺ signal triggered by glucagon.

	Materials and Methods

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Materials
Miniglucagon was obtained from Peninsula Laboratories Inc. Thapsigargin, caffeine, oligomycin, FCCP, penicillin-streptomycin solution, trypsin, nucleotides, and bovine serum albumin were purchased from Sigma Chemical Co. Fura 2, fura 2-AM, and pluronic acid F-127 were from Molecular Probes. Fetal calf serum was from GIBCO-BRL. PBS 2040 and medium 199 were obtained from Eurobio. Digitonin (Merck) had been recrystallized and stored as an 8 mg/mL solution in dimethyl sulfoxide. [⁴⁵Ca] (5 to 30 mCi/mg) and L-[4,5-³H]leucine (40 to 60 Ci/mmol) were from ICN. Isradipine was from Research Biochemical International. Japan paper was obtained from Sennelier. An IP₃ radioreceptor assay kit was purchased from Dupont-NEN.

Isolation of Chick Embryo Ventricular Cells
Fertile eggs were obtained from the Haas farm (Kaltenhouse, France). Primary monolayer cultured heart cells were prepared from 13-day-old chick embryo ventricles as previously described.^{6 15} The cells were dissociated by repeated cycles of trypsinization in sterile PBS 2040 medium containing 0.008% (wt/vol) trypsin. Dispersed cells were collected and diluted in ice-cold buffer A (medium 199 containing 0.1% [wt/vol] NaHCO₃, 0.01% [wt/vol] L-glutamine, and 0.1% penicillin-streptomycin antibiotic solution) supplemented with 20% (vol/vol) fetal calf serum to inactivate trypsin. The cells were then filtered through sterile japan paper and centrifuged at 150g for 10 minutes to wash out the trypsin. The supernatant was discarded, and the cells were suspended in buffer A and incubated for 1 hour in humidified 5% CO₂/95% air at 37°C in plastic dishes for adhesion of fibroblastic cells. Cells that remained in suspension were filtered again, and the cell suspension was diluted to 5 to 7x10⁵ cells per milliliter in buffer A. Cells in suspension were kept in buffer A, previously bubbled with 5% CO₂/95% air, at 4°C until used, up to 5 days.

Measurements of ⁴⁵Ca Accumulation Into Intracellular Compartments
Isolated myocytes (5x10⁵ cells per milliliter), suspended in buffer A supplemented with 5% (vol/vol) fetal calf serum, were plated on glass coverslips in multiwell plates and kept at 37°C in humidified 5% CO₂/95% air for 24 hours. As described by Marsh et al,¹⁶ before the experiment, the cells on glass coverslips were further incubated for 24 hours in buffer A containing 120 mg/L L-[4,5-³H(N)]leucine (1 µCi/mL) in order to label cell proteins. After washing in 2x1 mL saline buffer B (mmol/L: glucose 10, NaCl 130, KCl 5, HEPES 10 [buffered at pH 7.4 with Tris base], MgCl₂ 1, CaCl₂ 2), at time zero of the experiment, the cells were immersed in 1 mL of saline buffer B containing 2 mmol/L [⁴⁵Ca] (5 µCi/mL) in the presence or in the absence of 0.1 nmol/L miniglucagon and with or without 10 mmol/L caffeine. After various periods of incubation, the cells were washed two times for 10 seconds in 10 mL Ca²⁺-free saline buffer B at 25°C. In order to specifically evaluate the ⁴⁵Ca accumulation into the intracellular stores, the cells were next subjected to digitonin lysis, which selectively disrupts the SL membranes. This procedure has been described by Altschuld et al¹⁷ and consisted of an incubation for 45 seconds at 25°C in Ca²⁺-free saline buffer B supplemented with 0.1 mmol/L EGTA, 10 mmol/L MgCl₂, 10 mmol/L ATP, 5 µmol/L ruthenium red, and digitonin (16 µg per milligram protein). Mg²⁺-ATP was added to protect against hypercontracture and ruthenium red to block Ca²⁺ efflux from the SR. Intracellular Ca²⁺ accumulation was estimated from ⁴⁵Ca recovered into the digitonin-resistant structures attached on the coverslips after they were dissolved in 0.2 mol/L NaOH for 2 hours at room temperature. Determination of the ratio of [³H] counts to protein concentration and simultaneous counting of [³H] and [⁴⁵Ca] permitted normalization of Ca²⁺ uptake data per milligram cell protein. Data are expressed as mean±SEM. The significances of differences from the control values were analyzed by Student's t test.

Fura 2 Loading and Ca²⁺ Imaging
The cells were plated in plastic dishes, the bottom of which was replaced by a glass coverslip that had been coated with laminin (1 µg/mL), and were incubated at 37°C in humidified 5% CO₂/95% air for 17 to 24 hours. Cells, attached to laminin, were bathed in 2 mL of saline buffer B and were incubated for 20 minutes at 25°C with 1.5 µmol/L fura 2-AM (3 µL of 1 mmol/L fura 2-AM in dimethyl sulfoxide), in the presence of 1 mg/mL bovine serum albumin and 0.05% (w/vol) pluronic acid F-127 to improve fura 2 dispersion and facilitate cell loading. Cells were then washed with saline buffer B (twice, 2 mL) and allowed to incubate in the same buffer for 15 minutes at 25°C to facilitate hydrolysis of intracellular fura 2-AM. The concentration of fura 2 accumulated in the cells was estimated as described previously by Donnadieu et al.¹⁸ This consisted in comparing the fluorescence intensity of the cells loaded with fura 2-AM with that of optical oil droplets, similar in size to embryonic chick ventricular cells (0.9 to 1 µm) and loaded with known concentrations of fura 2, under the same experimental conditions (same camera gain setting, same attenuation filter). Under usual loading conditions, the average intracellular concentration of fura 2 was 15 µmol/L.

Ca²⁺ imaging, developed by A. Trautman in collaboration with the IMSTAR Co, was essentially described by Donnadieu et al.¹⁹ A Nikon Diaphot inverted microscope with epifluorescence was used. The light from a 100-W xenon lamp was filtered alternatively through either 350/380-nm or 360/380-nm filters to determine either [Ca²⁺]_i or the fluorescence ratio at 360 to 380 nm (F360/F380), respectively. Fura 2 fluorescence (Nikon UV-Fluor x40 objective) was filtered at 510 nm and recorded by an intensified CCD Photonic Science camera.

All imaging studies were performed on cells in which no spontaneous rise in [Ca²⁺]_i was observed before experimental manipulation. We used the term "quiescent" cells as opposed to "electrically stimulated" cells.

[Ca²⁺]_i determination was performed in quiescent cells perfused with saline buffer B, containing 2 mmol/L CaCl₂ and drugs and peptides as indicated. Each fluorescence image at 350 and 380 nm was the average of four images, in order to improve the signal-to-noise ratio, and one Ca²⁺ ratio image was recorded every 1.2 to 3 seconds. The [Ca²⁺]_i was calculated according to the formula of Grynkiewicz et al²⁰ :

(1)

where K_d is the dissociation constant of the fura 2–Ca²⁺ complex; ß is the ratio of the fluorescence of free and Ca²⁺-bound fura 2 measured at 380 nm and is related to the optical characteristics of the system; R is the ratio of the fluorescence signals measured at 350 nm (from which the background fluorescence at 350 nm is subtracted in the off-line analysis) and 380 nm (minus the background at 380 nm); and R_min and R_max are the limiting values of R in the presence of zero and saturating [Ca²⁺], respectively. For in vitro calibration, the value of R was determined in 14 different calibrating solutions (made in saline buffer B with various EGTA concentrations in order to vary free Ca²⁺ from 0.01 µmol/L to 2 mmol/L) in the presence of 15 µmol/L fura 2. Free Ca²⁺ concentrations of Ca²⁺/EGTA reference solutions were calculated by using the EQUIV program (Thierry Capiod, INSERM Unité 274, Orsay). An excellent linear fit of the data was obtained with Equation 1 for free Ca²⁺ between 0.01 and 1.5 µmol/L, which allowed, without ambiguity, the determination of K_dß. Depending on experimental settings, the values that we determined were as follows: R_min=0.3 to 0.4, R_max=8 to 13.5, and K_dß=1000 to 1700 nmol/L.

In vivo determinations of R_min and R_max were performed according to the protocol described by Pucéat et al.²¹ Maximal fluorescence was determined in the presence of 2 µmol/L 4-bromo-A23187, in cells that were previously bathed at 25°C for 1 hour in saline buffer solution containing 2 mmol/L CaCl₂, without glucose and with 3 mmol/L amytal and 5 µmol/L FCCP, in order to deplete cellular ATP and to avoid cell contractures or bursts. Minimal fluorescence was obtained by bathing cells for 1 hour in saline buffer devoid of CaCl₂ and containing 3.3 mmol/L EGTA with 2 µmol/L 4-bromo-A23187. R_max and R_min values were determined after subtraction of cell autofluorescence, measured in the presence of 1 mmol/L MnCl₂. Several studies have previously reported marked differences between the in vitro and in vivo calibration data.¹⁸ In the present study, the values determined in vivo for R_min (0.30 to 0.40) were equivalent to the in vitro values. In contrast, the R_max values determined in vivo approached 6 and thus differed somehow from the in vitro data. This is not surprising, since intracellular systems in living cells prevent high [Ca²⁺]_i rises. Nevertheless, in vivo calibrations gave a ratio of R_max to R_min of 20, which is an index of the dynamic range of our system that is comparable to the ranges usually reported in imaging systems, which vary from 12 to 25 (see Reference 19). Practically, [Ca²⁺]_i calculations always took into account in vivo R_min and R_max and in vitro K_dß.

In a series of experiments, cells were stimulated, and data were presented as the fluorescence ratio. Field electrical stimulation (square waves, 10-millisecond duration, amplitude 20% above threshold, 0.5 Hz) was supplied through a pair of platinum electrodes connected to the output of a HAMEG stimulator. Cells were perfused with saline buffer B containing 1.27 mmol/L CaCl₂ and stimulated until a steady state level of the intracellular Ca²⁺ transients was achieved, before each protocol, as previously described by Bassani et al.²² Drugs and peptides were added a few seconds after interruption of stimulation. The time resolution of the imaging system was increased by restricting the microscopic field with the aid of an adjustable window and by recording only fluorescence images at 380 nm every 0.3 second (average of two images). The fluorescence at 360 nm was recorded at time zero and at the end of the experiment to check for photobleaching. Data are presented as the fluorescence ratio F360/F380, calculated after subtracting respective backgrounds.

In all experiments, applications of the different compounds were performed by including them in the perfusion medium. All tracings of [Ca²⁺]_i or the fluorescence ratio are representative of at least 10 cells and were performed on at least two different cell isolations.

	Results

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Miniglucagon Increases ⁴⁵Ca Accumulation Into Caffeine-Sensitive SR Stores
Quiescent embryonic chick heart cells were incubated for various periods of time in a medium containing 2 mmol/L [⁴⁵Ca], in the absence or in the presence of 0.1 nmol/L miniglucagon. This concentration was chosen since it evoked a maximal effect of the peptide on heart cell contraction.⁶ At the end of the incubation period, myocytes were first washed with a Ca²⁺-free buffer and then subjected to digitonin lysis, which selectively disrupts the SL.¹⁷ This allowed for the specific quantification of ⁴⁵Ca content of digitonin-resistant structures, after elimination of the cell medium. As shown in Fig 1, in the absence of miniglucagon, digitonin-resistant myocyte structures were able to accumulate an appreciable amount of [⁴⁵Ca], which reached a steady state level within 1 minute. Exposing cells to miniglucagon resulted in a marked increase in the ⁴⁵Ca content of internal stores, which, after a 3- or 5-minute incubation, amounted to 142% and 180% over the control level, respectively. To determine whether miniglucagon action relied on the SR stores, we examined the effect of caffeine, which has previously been shown to increase the opening probability of the Ca²⁺-release channels in the SR compartment.²³ As shown in Fig 1, after a 5-minute incubation, caffeine alone elicited a 40% decrease compared with the control level in the ⁴⁵Ca content of intracellular stores. It may be noted that this gave an estimation of the caffeine-sensitive SR Ca²⁺ compartment versus the other intracellular Ca²⁺ pools, including mitochondria and nucleus. Added together with miniglucagon, caffeine completely prevented the accumulation of ⁴⁵Ca into intracellular stores elicited by the peptide. These results suggest that upon miniglucagon action, Ca²⁺ is accumulated into cardiomyocytes being sequestered in a caffeine-sensitive SR compartment.

View larger version (28K): [in this window] [in a new window]   Figure 1. Miniglucagon increases the accumulation of 45Ca into intracellular stores. At time zero, embryonic chick heart cells were exposed for the indicated periods of time to 2 mmol/L [45Ca] (5 µCi/mL) in the absence or in the presence of 0.1 nmol/L miniglucagon and with or without 10 mmol/L caffeine. Cells were thereafter submitted to digitonin lysis, and 45Ca accumulation in the intracellular stores was quantified. Data are expressed as the mean±SEM of 15 to 25 values obtained from two or three different experiments. *P<.05 and ***P<.001 vs the control values by Student's t test.

Miniglucagon Potentiates Caffeine-Induced Ca²⁺ Mobilization
The effect of miniglucagon on intracellular Ca²⁺ transients associated with caffeine contractures was examined in fura 2–loaded cells. Fig 2 shows intracellular Ca²⁺ transients during electrical stimulation and caffeine contractures obtained under steady state conditions, ie, within a few seconds after interruption of electrical stimulation, according to the protocol described by Bassani et al²² (also see "Materials and Methods"). As expected,²² the application of 10 mmol/L caffeine produced a unique intracellular Ca²⁺ transient, larger than those observed during electrical stimulation. Miniglucagon alone had no effect (Fig 5B). In contrast, the application of 10 mmol/L caffeine together with 0.1 nmol/L miniglucagon resulted in a train of intracellular Ca²⁺ transients (Fig 2B): a mean of 4±1 transients (n=36) was observed over a period of 40 seconds. It should also be noted that when normalized as a percentage of the control amplitude, defined as the mean amplitude of the intracellular Ca²⁺ transients during electrical stimulation, the amplitude of the intracellular Ca²⁺ transients after the combined application of caffeine and miniglucagon was higher than that of the single intracellular Ca²⁺ transient observed with caffeine alone (127±6% [n=36] and 92±6% [n=31] of the control amplitude, respectively). The L-type Ca²⁺ channel blocker isradipine²⁴ at 100 nmol/L, a concentration at which it totally abolished the intracellular Ca²⁺ transient during electrical stimulation (not shown), had no effect on the Ca²⁺ responses triggered by caffeine applied either alone or in combination with miniglucagon (Fig 2C and 2D). In the presence of isradipine, the application of 10 mmol/L caffeine alone produced a unique intracellular Ca²⁺ transient over a period of 40 seconds, with a mean amplitude of 96±7% (n=13) compared with the control amplitude (Fig 2C). Under the same conditions, the addition of miniglucagon together with caffeine triggered a mean of 4±1 intracellular Ca²⁺ transients (n=18) over a period of 40 seconds, with a mean amplitude of 111±3% (n=18) compared with the control amplitude (Fig 2D).

View larger version (27K): [in this window] [in a new window]   Figure 2. Miniglucagon potentiates Ca2+ release from the intracellular stores induced by caffeine. Cells were electrically stimulated at 0.5 Hz; stimulation was discontinued a few seconds before the addition of 10 mmol/L caffeine alone (A), 10 mmol/L caffeine plus 0.1 nmol/L miniglucagon (B), 10 mmol/L caffeine plus 100 nmol/L isradipine (C), or 10 mmol/L caffeine plus 0.1 nmol/L miniglucagon plus 100 nmol/L isradipine (D). These data were representative of at least 30 cells obtained from two different isolations.

View larger version (29K): [in this window] [in a new window]   Figure 5. Miniglucagon potentiates the cytosolic Ca2+ signals evoked by glucagon on cells electrically prestimulated. Cells were stimulated until a steady state level of the intracellular Ca2+ transients was achieved. Stimulation was then discontinued before the addition of 30 nmol/L glucagon (A), 0.1 nmol/L miniglucagon (B), or 30 nmol/L glucagon+0.1 nmol/L miniglucagon (C). These data are representative of at least 20 cells obtained from two different isolations.

Miniglucagon had no effect on IP₃ production in quiescent embryonic chick heart cells, in conditions in which 100 µmol/L acetylcholine evoked a threefold increase in the IP₃ level (from 20±6 to 68±22 pmol IP₃ per milligram protein after a 3-minute incubation time).

Taken together, these data suggest that miniglucagon action, which leads to a higher Ca²⁺ loading of caffeine-sensitive SR stores, does not rely either on Ca²⁺ influx through L-type Ca²⁺ channels or on IP₃-mediated Ca²⁺ mobilization.

We next examined the effect of miniglucagon in fura 2–loaded cells in which the SR Ca²⁺ storage capacity was reduced. This was achieved by pretreatment of quiescent cells with 1 µmol/L thapsigargin, a specific inhibitor of the SR Ca²⁺ pump.²⁵ Preincubation with thapsigargin for 30 minutes resulted in a significant increase in basal [Ca²⁺]_i (Fig 3A) (from 45±5 to 60±5 nmol/L [Ca²⁺]_i) and abolished completely the caffeine-induced Ca²⁺ signals (data not shown; see Reference 25), indicating SR Ca²⁺ depletion. The subsequent addition of miniglucagon caused a gradual increase in [Ca²⁺]_i (Fig 3A), which was not detected in the absence of thapsigargin (Fig 3B). This observation demonstrates that miniglucagon produces an accumulation of Ca²⁺ in the cell that is normally immediately compensated by its sequestration into the SR compartment via the SR Ca²⁺ pump. It should be noted that the increase in [Ca²⁺]_i elicited by miniglucagon in the presence of thapsigargin lasted for a few minutes only. In fact, substitution of NaCl in the extracellular medium by equimolar (130 mmol/L) LiCl prolonged the increase in [Ca²⁺]_i that was due to miniglucagon (Fig 3A), indicating that the return of [Ca²⁺]_i to the basal level was dependent on the Na⁺-Ca²⁺ exchanger activity.

View larger version (17K): [in this window] [in a new window]   Figure 3. Miniglucagon produces a rapid [Ca2+]i increase in quiescent cells treated with thapsigargin. The traces were recorded from individual quiescent cells. A, The cells were pretreated for 30 minutes with 1 µmol/L thapsigargin and maintained, at time zero, either in saline buffer B containing 130 mmol/L NaCl (lower trace) or incubated in saline buffer B in which NaCl had been replaced by 130 mmol/L LiCl (upper trace). When indicated, cells were exposed to 0.1 nmol/L miniglucagon for 3 minutes in the continual presence of 1 µmol/L thapsigargin. B, The cells were maintained in saline buffer B containing 130 mmol/L NaCl and exposed when indicated to 0.1 nmol/L miniglucagon. These data are representative of at least 28 cells obtained from four different isolations.

Miniglucagon Potentiates Glucagon-Induced Ca²⁺ Mobilization
Since, physiologically, one may expect that miniglucagon and glucagon act in concert, it was of interest to evaluate the combined effect of both peptides on [Ca²⁺]_i.

The first series of experiments was performed in conditions in which ⁴⁵Ca accumulation was observed upon miniglucagon action, ie, in cells maintained in resting conditions. The pattern of Ca²⁺ mobilization elicited by 30 nmol/L glucagon consisted of sporadic Ca²⁺ spikes (Fig 4A). This small mobilization of Ca²⁺ produced by glucagon was considerably potentiated by 0.1 nmol/L miniglucagon (Fig 4A). Note that miniglucagon alone had no effect (Fig 3B).

View larger version (27K): [in this window] [in a new window]   Figure 4. Miniglucagon potentiates the cytosolic Ca2+ signals evoked by glucagon or 8-bromo-cAMP on quiescent cells. During the periods indicated, the quiescent cells were successively exposed to 30 nmol/L glucagon and 30 nmol/L glucagon+0.1 nmol/L miniglucagon (A) or to 75 µmol/L 8-bromo-cAMP and 75 µmol/L 8-bromo-cAMP+0.1 nmol/L miniglucagon (B). These data are representative of 23 and 19 cells, respectively, obtained from two different isolations.

Application of glucagon to heart cells leads to an increase in cAMP; thus, we examined the effect of the permeant analogue 8-bromo-cAMP. The nucleotide, applied alone, reproduced the effect of glucagon, eliciting single spikes of [Ca²⁺]_i (Fig 4B). Furthermore, the combination of 8-bromo-cAMP with miniglucagon reproduced the train of Ca²⁺ transients promoted by glucagon plus miniglucagon (Fig 4B).

Since SR Ca²⁺ loading in quiescent cells is difficult to ascertain, a second series of experiments was performed by using the same protocol as previously described for caffeine, in which cells were prestimulated in order to ensure maximal loading of Ca²⁺ into the SR before application of glucagon, miniglucagon, or glucagon plus miniglucagon. Glucagon (30 nmol/L), perfused alone immediately after the interruption of electrical stimulation, produced a single Ca²⁺ transient over a 180-second period, the amplitude of which was 103±3% (n=29) of the control amplitude (Fig 5A). In the same conditions, miniglucagon alone, at 0.1 nmol/L, did not trigger any intracellular Ca²⁺ signal (Fig 5B). In contrast, the combination of 30 nmol/L glucagon with 0.1 nmol/L miniglucagon elicited a train of Ca²⁺ spikes (23±2 Ca²⁺ transients over a 180-second period, n=25) typical of CICR phenomenon, with a mean amplitude that was 140±5% of the control amplitude (Fig 5C). These observations confirmed those made on quiescent cells.

We have previously shown that in heart cells, phosphorylation of L-type Ca²⁺ channels occurs as a direct consequence of cAMP production by glucagon,⁹ leading to a Ca²⁺ influx that triggers Ca²⁺ release from the SR compartment.²⁶ Thus, to verify whether intracellular Ca²⁺ transients triggered by glucagon relied on Ca²⁺ influx via L-type Ca²⁺ channel activation, experiments were performed in which glucagon was perfused in the presence of isradipine. As shown in Fig 6A, when isradipine was perfused with glucagon, after interruption of electrical stimulation, no intracellular Ca²⁺ transient was detected. Isradipine also totally abolished the response elicited by the combination of glucagon plus miniglucagon (Fig 6B compared with Fig 5C). This result suggested that L-type Ca²⁺ channels are activated by glucagon, which may trigger the mobilization of intracellular Ca²⁺, presumably from the SR store.

View larger version (25K): [in this window] [in a new window]   Figure 6. Ca2+ mobilization by glucagon, in the absence or in the presence of miniglucagon, relies on the activation of isradipine-sensitive Ca2+ channels. Cells were stimulated until a steady state level of the intracellular Ca2+ transients was achieved. Stimulation was then discontinued before the addition of 30 nmol/L glucagon+100 nmol/L isradipine in the absence (A) or in the presence (B) of 0.1 nmol/L miniglucagon. These data are representative of 20 cells obtained from two different isolations.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
This report provides further insight into the mechanism of action of glucagon and of its metabolite, miniglucagon, in the heart. Combining ⁴⁵Ca uptake experiments with fura 2 imaging studies, we demonstrate a combined action of both peptides on cardiomyocyte Ca²⁺ homeostasis, which relies on (1) miniglucagon-induced Ca²⁺ loading of caffeine-sensitive SR Ca²⁺ stores and (2) the release of Ca²⁺ from these same stores, triggered by glucagon.

In quiescent cells, miniglucagon increased ⁴⁵Ca content into caffeine-sensitive stores (Fig 1). It is noteworthy that under our experimental conditions, an increase in ⁴⁵Ca content may represent either ⁴⁵Ca accumulation or increased ⁴⁵Ca exchange. In the present experiments, caffeine completely suppressed the miniglucagon-induced increase in cell ⁴⁵Ca loading. Since caffeine is known to trigger Ca²⁺ release from the SR compartment without affecting its uptake capacity, these results support the conclusion that miniglucagon produces ⁴⁵Ca accumulation into the SR compartment rather than accelerating its exchange.

In fura 2–loaded cells, within a few seconds after interruption of electrical stimulation, miniglucagon potentiated [Ca²⁺]_i mobilization induced upon caffeine contracture (Fig 3A). The absence of the intracellular Ca²⁺ transient upon application of the peptide on cells that were responsive to electrical stimulation indicated the inability of miniglucagon to promote, by itself, the release of Ca²⁺ from the SR and/or to evoke action potentials by depolarizing the membrane (Fig 5B). This supports the above conclusion that miniglucagon action resulted in Ca²⁺ accumulation into the SR stores (Figs 1 and 3A). Ca²⁺ accumulation into the SR stores evoked by miniglucagon occurred without a detectable modification of the average [Ca²⁺]_i (Fig 3B). In contrast, in the presence of thapsigargin, ie, in conditions in which the SR Ca²⁺ pump was inhibited, miniglucagon action resulted in a large increase in [Ca²⁺]_i (Fig 3A). These observations indicated that under normal conditions, the SR Ca²⁺ pump could prevent Ca²⁺ accumulation in the cytosolic compartment. The buffering capacity of the cardiac SR in face of trans-SL Ca²⁺ fluxes has been previously reported by Janczewski and Lakatta,²⁵ who demonstrated the rapid sequestration by the SR of at least 50% of the Ca²⁺ entering the cells during a single postrest stimulation of guinea pig ventricular myocytes.

It may be noted that in imaging studies, miniglucagon action seemed to be immediate (Figs 2, 4, and 5). In contrast, miniglucagon-induced ⁴⁵Ca accumulation could be detected only after 3 minutes of incubation (Fig 1). We interpret such differences as being due to a lower sensitivity of the isotopic technique.

On the basis of our previous reports, inhibition of the SL Ca²⁺ pump by miniglucagon could be responsible for Ca²⁺ accumulation in the cell.^{12 13 14} This hypothesis cannot be proved because of the lack of specific inhibitors of the SL Ca²⁺ pump; however, the long-term impairment of the SL Ca²⁺ pump, such as in diabetes,²⁷ in genetically linked cardiomyopathy,²⁸ or in septic shock,²⁹ has been implicated in the development of Ca²⁺ overload leading to myocardial dysfunction. Alternatively, the rise in intracellular Ca²⁺ triggered by miniglucagon could occur via a Ca²⁺ influx across the SL. However, the lack of effect of isradipine on the potentiation by miniglucagon of caffeine action (Fig 2) supports the hypothesis that the action of miniglucagon does not occur through L-type Ca²⁺ channels.

Glucagon, applied alone, induced sporadic Ca²⁺ transients, which were reproduced with 8-bromo-cAMP. Isradipine blocked the transients elicited in the presence of glucagon alone or glucagon plus miniglucagon. In a previous study, we showed that glucagon acted via a cAMP-dependent phosphorylation of the isradipine-sensitive L-type Ca²⁺ channels in cardiac cells.^{9 10} In addition, it has been reported that phosphorylation by the cAMP-dependent kinase of the cardiac SR CICR channel (or ryanodine receptor) leads to a more open state of the channel.³⁰ Taken together, these observations support the proposal that glucagon, via cAMP, elicits Ca²⁺ influx through L-type Ca²⁺ channels, triggering CICR, which is also facilitated by the simultaneous phosphorylation of the CICR channel but which relies on the filling state of the SR compartment.³¹

Our previous data demonstrated that activation of the cAMP pathway by glucagon was necessary but not sufficient to elicit contraction.⁶ Accordingly, whereas it is well established that ß-adrenergic agonists stimulate adenylyl cyclase activity, several reports have nevertheless demonstrated that there may be ß-adrenergic–mediated pathways for increasing myocardial inotropy independent of cAMP formation.^{32 33} Furthermore, stimulation of other receptor types in heart cells, such as prostaglandin E₁ receptors, which also induces an increase in cAMP, has no effect on contraction. Thus, the existence of cAMP pools not linked to Ca²⁺ and contractile regulation has been suggested.³⁴ The present data show that the action of glucagon is dependent on both the cAMP pathway and a cAMP-independent Ca²⁺ loading of the SR. It is noteworthy that glucagon through this dual mechanism of action is proving more efficient than ß-agonists in reversing profound myocardial depressions, in particular those induced by Ca²⁺ channel blocker toxicity.⁴

In conclusion, under physiological conditions, the positive inotropic effect of glucagon in the heart may be ascribed to the combined and distinct actions of glucagon itself, the "mother" molecule, and of miniglucagon, the "daughter" metabolite. These actions may be summarized as the ability of miniglucagon to accumulate Ca²⁺ into SR stores and that of glucagon to induce CICR from these stores. Preliminary experiments performed on electrically stimulated myocytes confirmed the synergistic efficiency of both peptides. Thus, continuous perfusion with 30 nmol/L glucagon alone evoked a limited increase in the amplitude of electrically stimulated intracellular Ca²⁺ transients (118±2% of control amplitude, n=41). In contrast, when cells were perfused with glucagon (30 nmol/L) plus miniglucagon (0.1 nmol/L), a marked rise in the amplitude of intracellular Ca²⁺ transients (197±8% of the control amplitude, n=52) was observed.

	Selected Abbreviations and Acronyms

 

ß = fluorescence ratio of free to Ca2+-bound fura 2 CICR = Ca2+-induced Ca2+ release FCCP = carbonyl cyanide p-(trifluoromethoxy)phenyl-hydrazone IP3 = inositol tris-phosphate Kd = dissociation constant of the fura 2–Ca2+ complex Rmin and Rmax = limiting values of the fluorescence ratio in zero (min) and saturating (max) [Ca2+] SL = sarcolemma, sarcolemmal SR = sarcoplasmic reticulum, sarcoplasmic reticular

	Acknowledgments

 
This study was supported by the Institut National de la Santé et de la Recherche Médicale, the French Ministère de la Recherche et de la Technologie, and the Unité de Formation et de Recherche de Médecine, Créteil, Paris-Val de Marne. This material is also based on work supported by the North Atlantic Treaty Organization under a grant awarded in 1994. We thank M. Aggerbeck, R. Barouki, B. Crozatier, and R. Ventura-Clapier for helpful discussion and J. Hanoune for his permanent support.

Received February 21, 1995; accepted October 2, 1995.

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