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Am J Physiol Heart Circ Physiol 290: H1842-H1847, 2006. First published December 3, 2005; doi:10.1152/ajpheart.00388.2005
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Gs and Gi coupling of adrenomedullin in adult rat ventricular myocytes

Shivani Mittra and Jean-Pierre Bourreau

Department of Physiology, Institute of Cardiovascular Sciences and Medicine, The University of Hong Kong, Hong Kong, Special Administrative Region of China

Submitted 20 April 2005 ; accepted in final form 17 November 2005


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
Adrenomedullin (ADM) acts as an autocrine or a paracrine factor in the regulation of cardiac function. The intracellular mechanisms involved in the direct effect of ADM on adult rat ventricular myocytes (ARVMs) are still to be elucidated. In ARVMs from normal rats, ADM produced an initial (<30 min) increase in cell shortening and Ca2+ transients and a marked decrease in both on prolonged incubation (>1 h). Both effects were sensitive to ADM antagonist ADM-(22–52). Treatment with SQ-22536, an inhibitor of adenylate cyclase, blocked the positive inotropic effect of ADM and potentiated its negative inotropic effect. The negative inotropic effect was sensitive to inhibition by pertussis toxin (PTX), an inhibitor of Gi proteins and KT-5720, an inhibitor of PKA. The observations suggest a switch from Gs-coupled to PTX-sensitive, PKA-dependent Gi coupling by ADM in ARVMs. The ADM-mediated Gi-signaling system involves cAMP-dependent pathways because SQ-22536 further increased the negative inotropic actions of ADM. Also, because ADM is overproduced by ARVMs in our rat model of septic shock, ARVMs from LPS-treated rats were subjected to treatment with ADM-(22–52) and PTX. The decrease in cell shortening and Ca2+ transients in LPS-treated ARVMs could be reversed back with ADM-(22–52) and PTX. This indicates that ADM plays a role in mediating the negative inotropic effect in LPS-treated ARVM through the activation of Gi signaling. This study delineates the intracellular pathways involved in ADM-mediated direct inotropic effects on ARVMs and also suggests a role of ADM in sepsis.

cell shortening; calcium transient; pertussis toxin; adenosine 3'5'-cyclic monophosphate-dependent protein kinase inhibitor; septic shock

ADRENOMEDULLIN [ADM-(1–52)], first isolated from human pheochromocytoma, is a multifunctional regulatory peptide (16). Circulating ADM levels have been observed to be upregulated under various pathophysiological conditions, with particularly high levels evident in septic shock (6, 22). ADM immunoreactivity, expression of mRNA for ADM, and abundant binding sites have been detected in heart where it is regarded as an autocrine or paracrine factor regulating the cardiac function (14). Adult rat ventricular myocytes (ARVMs) and nonmyocytes express ADM and the protein subunits known to constitute the functional ADM receptors, namely, calcitonin receptor-like receptor (CRLR)/receptor activity-modifying protein (RAMP)2 (ADM1) and CRLR/RAMP3 (ADM2) heterodimers, and a concerted upregulation of ADM, RAMP2, and RAMP3 mRNA levels in cardiomyocytes isolated from rats with heart failure has been reported (21).

Generally looked on and reported as a compensatory peptide in cardiac functioning, the direct inotropic effects of ADM at the cardiac level are still inconclusive. Positive inotropy (11), negative inotropy (12, 19, 24, 10, 18), and no inotropy (20, 27) have been attributed to it. The major signal transduction pathway activated by ADM appears to be Gs-mediated adenylate cyclase (AC)/cAMP system (11). However, not all effects of ADM can be explained by the cAMP/PKA pathway (5). In our laboratory, a dual inotropic effect to ADM, specific to inhibition by its antagonist ADM-(22–52), has been observed in adult rat ventricular myocytes (10, 18). ADM was observed to produce an initial (on <30 min incubation) increase in cell shortening and Ca2+ transients and, on prolonged incubation (>1 h), a marked decrease in cell shortening and Ca2+ transients. ADM being a G protein-coupled receptor has been shown to undergo desensitization in rat mesangial cells (23) and downregulation in rat vascular smooth muscle cells (13). In this study we have tried to delineate the intracellular pathways involved in the direct inotropic effects of ADM on ARVM.

Because ADM is excessively produced in septic shock, we simulated its overproduction by incubating normal ventricular myocytes with a high dose of ADM for a long time to compare the negative inotropic effect of ADM with that produced in myocytes isolated from LPS-treated rats.


   MATERIALS AND METHODS
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 MATERIALS AND METHODS
RESULTS
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The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publications No. 85-23, Revised 1996). All animal experiments were approved by the Committee on the Use of Live Animals in Teaching and Research of the University of Hong Kong.

Animal models of septic shock. Endotoxemia was induced in male Sprague-Dawley rats, aged 10–15 wk and weighing 250 to 300 g, by an intraperitoneal injection of lipopolysaccharide (10 mg/kg) as previously described (26). Observations of conscious rats after LPS treatment were characteristic of endotoxemia, i.e., piloerection, apathy, and diarrhea. The rats (10%) that did not exhibit these characteristics were taken as LPS-resistant rats and consequently were not used for this study. The sham-operated control rats were injected with saline (1 ml/kg ip). AVRMs were isolated 4 h after LPS injection to be further treated with drugs.

Cell isolation procedure. Calcium concentration and cell shortening measurements were performed on freshly isolated ventricular myocytes. Enzymatically isolated ventricular myocytes were obtained by using the procedure previously described (26). After calcium reconstitution, the yield of viable myocytes was 45–50%. The final cell suspension contained a negligible amount of nonmyocytes. Cells were maintained in a Ca2+-containing HEPES-buffered solution (HBS) consisting of (in mM) 130 NaCl, 5.6 KCl, 1.25 CaCl2, 1 MgCl2, 10 glucose, 4 NaHCO3, and 10 HEPES (pH 7.4). The cells were used within 6 h of isolation. The average resting cell length for normal control cells was 116.54 ± 3.29 µm (n = 70 cells), and for LPS-treated cells, 112.0 ± 1.7 µm (n = 83 cells), and the difference was nonsignificant. In all experiments a cell length of >100 µm was chosen as a prerequisite for cell selection. The intracellular Ca2+ measurements were done at 24–26°C, and the myocyte contraction was observed at 32°C.

Intracellular calcium measurements. Changes in intracellular calcium concentration ([Ca2+]i) of paced ventricular myocytes were monitored fluorimetrically using the Ca2+-sensitive probe fura-2 (1 µM). The recording system included a Zeiss, Axiovert S170 inverted microscope fitted with epifluorescence (monochromator Till Polychrome II). The cells were alternatively illuminated at 340 and 380 nm at a frequency of 140 Hz. Emission for each excitation wavelength was filtered at 510 nm and digitized using interface (EPC9, HEKA). The 340-to-380 ratio was displayed online using the Xchart software. Steady-state responses of ventricular myocytes to electrical-field stimulation under control and test conditions were recorded from different cells. Cells were exposed to ADM by incubation in HBS containing an ADM concentration (100 nM or 1 nM) for a duration of 30 min and over 1 h, whereas control cells remained in normal HBS. Only rod-shaped myocytes with clear edges, no spontaneous contraction, and a resting 340-to-380 ratio <1 were selected.

Measurement of myocyte contraction. Contractile parameters of ventricular myoctes were assessed by a video-based edge-detection system (IonOptix, Milton, MA), which sampled cell length at 60 Hz. Briefly, the cells were placed in a chamber mounted on the stage of an inverted microscope (Nikon). The cells were field stimulated (Grass S88) with 20% suprathreshold voltage at a frequency of 0.2 Hertz (5-ms duration) with a platinum electrode. The myocyte being studied was displayed on the computer monitor with the help of an IonOptix MyoCam CCD camera, which was attached to the sidearm of the microscope. SoftEdge aqusition software (IonOptix) captured and converted the changes in cell length to digital signals. The signals were analyzed by IonWizard analysis software (IonOptix) to obtain the contractile parameters. Cell shortening was expressed as the percentage of resting cell length. The cells were incubated in different media (control and media plus drugs) for at least 30 min before stimulation. Data were recorded in steady-state conditions in each experimental medium. All the inhibitors tested had no effect per se (data not shown).

Hemodynamic measurements. Rats were anesthetized by an intraperitoneal injection of urethane (1.2 g/kg), a rectal probe was inserted, and body temperature was maintained at 37°C. The trachea was cannulated to facilitate respiration. The right femoral artery was cannulated and connected to a blood pressure transducer (MLT1050, ADInstruments). The signal for systemic arterial blood pressure was recorded in a personal computer via an analog-to-digital converter (PowerLab/410, ADInstruments). After a period of stabilization (~30 min), either LPS (10 mg/kg) or saline (1 ml/kg) was injected intraperitoneally. Blood pressure was monitored for 6 h. The animals were under the effect of urethane anesthesia for the entire experimental time.

Procedure for measuring mRNA levels. Four hours after intraperitoneal injection of LPS or saline, rats were euthanized and cardiac ventricles were dissected out and immediately frozen in dry ice and stored at –70°C until use. Frozen tissues were pulverized in liquid nitrogen before extraction. Tissues weighing 100–200 mg were homogenized in 2 ml of Trizol reagent (GIBCO-BRL, Life Technologies) using a polytron and were further processed according to the protocol as detailed previously (7). The details of the solution hybridization assay have been reported (9). The plasmids containing cDNA for AM (613 bp in length) and beta-actin (387 bp in length) were used to make RNA probes and standards. Standards or the tissue samples (5 µg total RNA) were incubated with 100,000 counts/min of 32P-labeled AM RNA riboprobe or 32P-labeled beta-actin RNA probe in hybridization buffer [80% formamide, 40 mM PIPES (pH 6.7), 400 mM NaCl, and 1 mM EDTA (pH 8.0)] at 45°C overnight, after preincubation at 85°C for 5 min. After RNase A and T1 digestion, the RNA hybrids were subjected to 4% PAGE (19:1 acrylamide to bis-acrylamide; USB, Cleveland, OH) at 180 V for ~1 h. The hybrids were visualized by exposure to an X-ray film (Super RX 20 x 25 cm, Fuji) at –70°C in a cassette with intensifying screens for at least 3 days. The hybrid bands on the gel were cut out, and their radioactivities were counted by a liquid scintillation counter (Beckman LS 6500, Multipurpose scintillation counter).

ADM assay. Rat ventricles were homogenized in 1 N acetic acid, and the mixture was boiled for 10 min in a water bath to inactivate the proteases. A 50-µl aliquot was taken for protein assay, and the remaining homogenates were centrifuged (Beckman AJ-21) at 13,000 rpm for 20 min at 4°C. The supernatants were lyophilized overnight and stored at –20°C. The lyophilized samples were reconstituted in assay buffer for the determination of immunoreactive (ir)-AM concentration as described previously (8, 9). Rat AM, AM antiserum, and 125I-labeled AM were purchased from Peninsula (Belmont, CA).

Data analysis. The response of each cell to stimulation was determined by averaging 10 successive transients in steady-state condition. Ca2+ transients are expressed as percent changes from resting 340-to-380 ratio in each cell or as {Delta}340-to-380 ratio (% control). Cell shortening is expressed as percent change in resting cell length. Results are expressed as means ± SE of n cells. Differences between two group means were evaluated by unpaired Student's t-test. Comparisons between multiple groups were made by one-way ANOVA with post hoc Newman-Keuls t-test. P < 0.05 was considered significant.

Materials. Fura-2 AM was purchased from Molecular Probes (Eugene, OR). Human ADM-(1–52) and human ADM-(22–52) and anti-ADM-(1–50) rat IgG were from Penynsula Laboratories (San Carlos, CA). SQ-22536, KT-5720, pertussis toxin (PTX), lipopolysaccharide Escherichia coli serotype 0111:B4, MEM, collagenase (type 1), BSA, and all other chemicals were from Sigma-Aldrich (St. Louis, MO).


   RESULTS
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DISCUSSION
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Myocytes from control rats. Within 30 min, pretreatment with ADM-(1–52) induced a significant increase in Ca2+ signal induced by field stimulation (Fig. 1, A and C). Pretreatment with ADM-(1–52) (100 nM for up to 1 h) did not significantly change the resting value of the 340-to-380 ratio (0.62 ± 0.02, n = 55 cells, vs. 0.65 ± 0.02, n = 55 cells). At more than 1 h, pretreatment with 100 nM ADM-(1–52) induced a significant decrease in calcium response (Fig. 1, B and C). A corresponding increase in cell shortening was observed on pretreatment with 1 nM ADM within 30 min, and a marked decrease in cell shortening was observed at >1 h (Fig. 2). Both the positive and negative inotropic responses were observed to be abolished by 100 nM ADM-(22–52), an antagonist of ADM-(1–52) in our earlier study (11).


Figure 1
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  		Fig. 1. Positive (A) and negative (B) effect induced by adrenomedullin (ADM)-(1–52) on Ca2+ transients recorded in control myocytes after incubation of cells for 30 min (A) or >1 h (B) in PSS with (bullet) or without ({circ}) 100 nM ADM-(1–52). 340/380, 340-to-380 nm excitation wavelength ratio. Data are means ± SE of 15 to 55 cells from 4–6 rats. C: effect of 100 µM SQ-22536 on 100 nM ADM-(1–52) and 10 nM isoproterenol (Iso)-mediated changes in Ca2+ transient in myocytes isolated from control rats. Data are means ± SE of 10 to 33 cells from 4–6 rats. *P < 0.05 vs. control; #P < 0.05 vs. ADM (<30-min group); $P < 0.05 vs. ADM (>1-h group); @P < 0.05 vs. Iso.

 

Figure 2
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  		Fig. 2. Effect of 4-h incubation with 2 µg/ml pertussis toxin (PTX) (A and B) and effect of 10 µM KT-5720 (C and D) on 1 nM ADM-(1–52)-mediated effects on electrically stimulated cell shortening of rat cardiomyocytes. Data are means ± SE of 14 to 52 cells from 4 to 6 rats. *P < 0.05 vs. control; $P < 0.05 vs. ADM (30 min); #P < 0.05 vs. ADM (90 min).

 
Pretreatment with SQ-22536 (100 µM, 30 min) completely abolished the increase in Ca2+ transient induced by 100 nM ADM-(1–52) (Fig. 1C). SQ-22536 further potentiated the decrease in Ca2+ transients observed with ADM-(1–52) incubation for >1 h (Fig. 1C). In our experimental conditions, SQ-22536 fully inhibited the increase in Ca2+ transients induced by 10 nM isoproterenol (Fig. 1C).

Regarding the pretreatment with PTX, incubating the ARVMs with 2 µg/ml PTX for 4 h abolished the decrease in cell shortening observed with pretreatment with ADM-(1–52) for >1 h while having no effect, per se, on the myocyte contractility (Fig. 2, A and B).

Regarding the pretreatment with KT-5720, incubating the ARVMs with KT-5720 (10 µM for 30 min) partially but significantly abolished the increase in cell shortening observed on pretreatment with ADM-(1–52) for <30 min. KT-5720 completely abolished the decrease in cell shortening observed on pretreatment with ADM >1 h (Fig. 2, C and D). KT-5720, per se, had no effect on the contractility of ARVMs (Fig. 2, C and D).

Effect of LPS injection on mean arterial blood pressure in anesthetized rats. Rats were randomly distributed in two groups of eight animals. Animals were anesthetized, the right femoral artery cannulated for blood pressure measurement. The baseline values of mean arterial blood pressure (MAP) before saline or LPS injection were not significantly different between the two groups of animals. Usually, LPS injection caused a transient fall in MAP, which returned to or above baseline within 2 h. MAP then gradually fell over the next 4 h (data not shown). Figure 3A shows that in rats injected with LPS (10 mg/kg ip), the MAP was significantly lower than sham-operated animals at the time when animals were euthanized for myocytes isolation, 4 h after LPS injection.


Figure 3
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  		Fig. 3. A: effect of LPS (10 mg/kg ip) on mean arterial blood pressure at 4 h after injection. B: ADM immunoreactivity in ventricles isolated 4 h after LPS injection. C: mean level of prepro-ADM mRNA in ventricles isolated 4 h after injection of LPS. Inset: X-ray film of PAGE of the representative ventricular RNA hybrids of prepro-AM and beta-actin mRNA at 4 h after LPS treatment. Upper bands (a), prepro-AM mRNA (613 bp); lower bands (b), beta-actin mRNA (387 bp). Samples from LPS-injected rats are in lanes 1, 3, and 5, whereas samples from control rats are in lanes 2 and 4. Data are means ± SE; n = 5 rats in each set. **P < 0.01, significantly different from sham-operated control.

 
Myocytes from LPS-treated rats. The ventricular ir-AM levels and prepro-AM mRNA levels were increased at 4 h after LPS treatment (Fig. 3, B and C). In ventricular myocytes isolated from rats euthanized 4 h after LPS injection, the resting [Ca2+] ratio value was significantly decreased compared with the normal myocytes (0.543 ± 0.01, n = 99 cells, vs. 0.61 ± 0.01, n = 154 cells in control myocytes, P < 0.05). The amplitude of the calcium transient elicited in myocytes isolated from LPS-treated rats by electrical-field stimulation was significantly decreased compared with transients obtained in myocytes isolated from control rats (Fig. 4A). In cardiomyocytes from LPS-treated rats, application of ADM-(1–52) had no effects on Ca2+ transient (data not shown). In contrast, 100 nM ADM-(22–52), an ADM receptor antagonist, induced a significant increase in calcium transient (Fig. 4A). Treatment of LPS-treated cells with PTX (2 µg/ml, 4 h) also induced a significant increase in calcium transients (Fig. 4B).


Figure 4
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  		Fig. 4. A: averaged Ca2+ transients recorded in myocytes isolated from sham-operated controls, LPS-treated rats, and LPS-treated + 100 nM ADM-(22–52) groups. B: averaged Ca2+ transients recorded in myocytes isolated from LPS-treated rats and LPS-treated + 2 µg/ml PTX groups. Data points represent means ± SE of 25 to 54 cells from 4 to 6 rats. C: cell shortening in electrically stimulated rat ventricular myocytes from control and LPS-treated rats. Effect of 2 µg/ml PTX, 100 nM ADM-(22–52), and 300 nM anti-ADM IgG on cell shortening in the LPS-treated group. Data are means ± SE of 20 to 60 cells from 4 to 6 rats. *P < 0.05 vs. normal control; #P < 0.05 vs. LPS-treated group.

 
In LPS-treated cells, the cell shortening was markedly decreased (Fig. 4C), and incubation with 100 nM of ADM-(22–52) or 300 nM of anti-ADM rat IgG restored the cell contractility. Treatment of LPS-treated cells with PTX (2 µg/ml, 4 h) markedly increased the contractility of shocked ARVMs back to the normal levels.


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study ADM-(1–52) was observed to stimulate a dual signaling pathway in isolated adult rat ventricular myocytes through the activation of an ADM-(22–52)-sensitive receptor. A marked increase in the cell shortening was observed within 30 min of incubation of ADM accompanied by an increase in Ca2+ transient. On prolonged exposure of >1 h, the positive inotropic effect was no longer observed. Rather, a marked negative inotropic effect was observed, accompanied by a decrease in Ca2+ transient. The ADM-mediated effects did not result from the activation of CGRP receptors, because CGRP inhibitor CGRP-(8–37) was unable to prevent ADM-(1–52)-induced changes in Ca2+ response and cell contractility, whereas both the effects were prevented by the ADM antagonist ADM-(22–52) in our earlier study (10). Both the change in cell contraction and Ca2+ transients was observed to be time dependent and dose independent (tested for ADM at 1 nM, 10 nM, and 100 nM; unpublished observations).

To investigate the intracellular mechanisms of ADM, the ARVMs were treated with SQ-22536, a potent inhibitor of AC. Pretreatment with SQ-22536 did not change the fluorescence resting value or the Ca2+ transient in response to electrical stimulation. It fully inhibited the increase in Ca2+ transients induced by 10 nM isoproterenol. SQ-22536 completely inhibited the increase in Ca2+ transients due to ADM-(1–52), indicating that the positive inotropic effect was a result of activation of an AC/cAMP-dependent pathway as extensively reported before (11, 25). KT-5720, an inhibitor of PKA, partially but significantly abolished the positive inotropy observed with ADM at <30 min, implicating activation of Gs/AC/cAMP/PKA pathway. Because KT-5720 could not fully inhibit the positive inotropic effect of ADM, it may also be stimulating cAMP-independent mechanisms, such as intracellular Ca2+ release, activation of PKC, and Ca2+ influx through L-type Ca2+ channel current (ICa,L) in ARVMs (27). ADM is documented to act through both PKA-dependent and -independent pathways in ARVMs. Recent reports (21) have observed a twofold increase in AC activity on stimulation with ADM in rat and cardiomyocytes. Our results indicate that ADM has positive inotropic effect on rat ventricular myocytes, at least partially through a cAMP-dependent pathway, which has also been observed by Ihara et al. (11). The observation that KT-5720 increases cell shortening nearly to the ADM at 30 min-induced levels and not to the control levels implicates that cAMP-dependent pathway may not be the only one contributing toward positive inotropy.

On prolonged incubation, when ADM induced a negative effect, pretreatment with SQ-22536 led to a further decrease in Ca2+ transient, suggesting that ADM may be activating two intracellular pathways, one leading to an AC-dependent positive inotropic effect and the other to an AC-dependent negative inotropic effect. ADM-mediated negative inotropic effects observed in rabbit ventricular myocytes were accompanied by a decrease in Ca2+ transient and ICa,L and were abolished by pretreatment with the nitric oxide (NO) synthase inhibitor N{omega}-monomethyl-L-arginine. In these cells, ADM was seen to significantly increase the intracellular cGMP content but not cAMP (12). However, in a previous study (10) 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a specific inhibitor of guanylate cyclase, did not affect the ADM-mediated effects. Moreover, pretreatment with N{omega}-nitro-L-arginine methyl ester was without any effect on the ADM-mediated actions, suggesting that the negative inotropic effect of ADM is through a NO/cGMP-independent pathway in adult rat ventricular myocytes (10). In the adult rat ventricular myocytes, the ADM-mediated Gi-signaling system involves cAMP-dependent pathways, because SQ-22536 further increased the negative inotropic actions of ADM.

The ADM-mediated decrease in cell shortening was completely reverted back by incubation with PTX. Moreover, the negative inotropic effect could be inhibited by KT-5720, a cell permeable inhibitor of PKA. This indicates that the negative inotropic effect was mediated by PTX-sensitive G proteins and that the activation of this pathway by ADM was dependent on PKA. Collectively, these data indicate that ADM, by coupling to different G proteins, may activate two different signaling cascades (Fig. 5). Examples of such dual coupling of beta-adrenergic receptor (beta-AR) and prostacyclin to Gs and Gi pathways have been reported (2, 3, 17, 29). There is growing awareness that in addition to the classical desensitization, there can be a simultaneous activation of another secondary signaling cascade. Agonist-mediated receptor phosphorylation has been reported to trigger molecular switching, whereby receptor alters its coupling from one G protein/effector system to a different G protein/effector system (3). beta2-ARs are reported to switch coupling from G{alpha}s to G{alpha}i in a mechanism involving initial G{alpha}s activation of AC and subsequent PKA phosphorylation; the phosphorylated beta2-AR, in turn, exhibits diminished coupling to Gs and increasing coupling to Gi in cardiac myocytes (2, 3, 29). PTX treatment of cardiac myocytes enhanced beta2-AR-stimulated increases in Ca2+ current, Ca2+ transients, and contraction amplitude (28). Evidence for a beta2-AR/Gi link has been observed in normal ARVMs, human atria, human ventricles, and failing rat heart (2, 4, 15, 29). Our results also show that the coupling of ADM to Gs switches over to Gi in a regulated manner because ADM receptor has to be phosphorylated by PKA before the PTX- sensitive Gi coupling can take place (Fig. 5). Because no effect of PTX was observed on ADM-mediated positive inotropy (unpublished observation), it suggested that ADM did not simultaneously act through activation of Gs and Gi receptor populations and that a switch from Gs to Gi coupling is taking place.


Figure 5
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  		Fig. 5. ADM, through ADM receptor, stimulates Gs-mediated activation of adenylate cyclase (AC) that leads to an increase in cAMP and PKA activation, resulting in a positive inotropic effect within 30 mins. Thereafter, activated PKA phosphorylates ADM receptor, favoring its coupling to Gi, producing an AC-mediated negative inotropic effect at >1 h incubation. The PTX-sensitive coupling to Gi can be inhibited by KT-5720, implicating PKA phosphorylation as critical for the Gs-to-Gi switch over.

 
In LPS-treated rats, the ARVMs showed a marked decrease in Ca transients and myocyte contractility, as previously shown in our laboratory (10, 18). The decrease in myocyte contraction reverted back by incubation with ADM antagonist and ADM antiserum. The changes in cell kinetics of shocked cells were also observed to normalize on treatment with ADM antagonist and ADM antiserum (18). This implicates a role of ADM in LPS-mediated septic shock. A significant increase in levels of ADM has been observed in plasma, ventricles, and isolated ventricular myocytes 4 h after the injection of bacterial endotoxin (LPS) in our earlier study, as described previously (18, 30). In the present study, a significant upregulation of ADM mRNA and immunoreactivity has been observed in rat heart at the same time point. Also, treatment with PTX reverted back the decrease in myocyte contraction and calcium transients, showing the role of Gi coupling-mediated propagation of negative inotropy in shocked cells. The PTX-sensitive, Gi-mediated loss of contractility in sepsis can be the cumulative effect of simultaneous Gi activation by many receptor types, beta-AR and ADM being among the contributors (1).

In conclusion, a switch from Gs coupling to PKA-dependent Gi coupling was observed with ADM in ARVMs, resulting in a shift from positive inotropy to negative inotropy, which was time dependent and dose independent.


   GRANTS
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 ABSTRACT
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This work was supported by a grant from the Committee on Research and a Conference Grant from the University of Hong Kong.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J.-P. Bourreau, Dept. of Physiology, 4/F, Laboratory Block, Faculty of Medicine Bldg., The Univ. of Hong Kong, 21 Sassoon Rd., Hong Kong SAR, China (e-mail: bourreau{at}hkucc.hku.hk)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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