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(Circulation Research. 1995;77:973.)
© 1995 American Heart Association, Inc.

Articles

Differential Regulation of Circulating Mg²⁺ in the Rat by ß₁- and ß₂-Adrenergic Receptor Stimulation

D. Keenan, A. Romani, A. Scarpa

From the Departments of Physiology and Biophysics (D.K., A.R., A.S.) and Surgery (D.K.), Case Western Reserve University, School of Medicine, Cleveland, Ohio.

	Abstract

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Abstract Extracellular Mg²⁺ homeostasis was studied in vivo in the anesthetized rat. Animals were infused with isoproterenol (ISO) for 10 minutes, and serum Mg²⁺ was measured after the infusion and then 10 and 20 minutes later. A dose-dependent increase in circulating Mg²⁺ was observed in animals infused with ISO at a rate of 0.1 µg · kg^-1 · min^-1 or higher. The time course of the effect demonstrated that circulating Mg²⁺ continued to increase 20 minutes after the end of the ISO infusion. A predicted maximal increase in serum Mg²⁺ concentration of 19.3% was derived with a predicted EC₅₀ of 0.08 µg · kg^-1 · min^-1. The maximal percent increase corresponded to a net increase of 6.7 µmol/300 g body wt. Because infusion of ISO resulted in changes in hemodynamic parameters, most notably a drop in blood pressure, a group of animals was infused with nitroprusside to mimic the hypotensive response via a nonadrenergic mechanism. Under these conditions, there was a transient increase in circulating Mg²⁺ that was largely inhibited by propranolol, indicating that hypotension per se was not responsible for the mobilization of Mg²⁺. Infusion of salbutamol, but not prenalterol, also induced an increase in circulating Mg²⁺. Pretreatment with butoxamine, ICI-118551, or propranolol prevented the ISO-induced increase in serum Mg²⁺. Pretreatment with atenolol minimally affected the ISO-induced changes in circulating Mg²⁺. Pretreatment with CGP-20271A actually enhanced the ISO-induced increase in circulating Mg²⁺. This evidence demonstrates the existence of a pool of Mg²⁺ that is mobilized into the circulation in response to selective ß₂-adrenergic stimulation.

Key Words: Mg²⁺ • circulation • ß-adrenergic stimulation • rats

	Introduction

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A large number of whole-body and cellular functions require Mg²⁺ and are influenced by changes in the concentration of free Mg²⁺ (see References 1 through 3^{1 2 3} for review). The relevance of these observations to the physiology of intact organisms is supported by a significant collection of data obtained from isolated cells and/or perfused organs that characterize the movement of Mg²⁺ into or out of mammalian cells. Although one group has recently failed to demonstrate an extrusion of Mg²⁺ from isolated rat cardiac myocytes in response to adrenergic stimulation,⁴ the consensus of the literature supports the observations that in response to physiological stimuli,^{5 6 7} Mg²⁺ can rapidly redistribute across the plasma membrane of liver cells,^{5 6 8 9} cardiac cells,^{7 9 10 11} erythrocytes,¹² thymocytes,¹³ pancreatic Beta cells,^{14 15} and sublingual acini.^{16 17} If such a large Mg²⁺ movement were to occur in vivo, overall Mg²⁺ homeostasis would be affected at three distinct levels. First, the concentration of Mg²⁺ in the cytoplasm, or within intracellular organelles, may be altered. Changes in the free Mg²⁺ would then be dependent on the Mg²⁺-buffering capacity present. Second, movement of Mg²⁺ across the plasma membrane into or out of cells would result in an opposite change in Mg²⁺ content in the extracellular space/plasma. Thus, Mg²⁺ could affect physiological processes in tissues different from those in which the movement primarily occurred. Third, changes in circulating Mg²⁺ would result in a concomitant modification of the amount of cation filtered in the kidney, which contributes significantly to the regulation of whole-body Mg²⁺ content.

The in vitro studies mentioned above suggest that adrenergic stimulation plays an important role in regulating cellular Mg²⁺ content. Previous studies performed in vivo have provided conflicting results regarding the effect of adrenergic stimulation on circulating Mg²⁺. The nonconsistent observations may in part be the result of the differences in the experimental protocols used, in terms of animal species, choice of agonist, and dose or duration of agonist used. The present study investigates the effect of short-term infusion of ß-adrenergic agonists at various concentrations on circulating Mg²⁺ levels in the anesthetized rat under well-defined protocols, which include accurate and simultaneous measurements of blood pressure, HR, body temperature, and Hct.

	Materials and Methods

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Infusion Method
Male Sprague-Dawley rats (250 to 350 g) were maintained on a standard Lab Chow diet (Purina) with alternating 12-hour day/night cycles in the Animal Resource Center at Case Western Reserve University.

The experimental technique used for all protocols is depicted in Fig 1. Surgical anesthesia was established with the subcutaneous injection of urethane (1 g/kg). Polyethylene catheters (PE-50, Becton-Dickinson) were placed in the tail artery, femoral artery, and femoral vein and flushed with 0.2 mL heparinized (20 U/mL) saline solution. The femoral artery catheter was connected to a blood pressure transducer (Statham), which was coupled to a monitor (Electronics for Medicine) to continuously display blood pressure and HR. Core body temperature was monitored with a rectal thermometer and maintained at 37°C with a heating blanket (Scientific and Research Instruments). After 30 minutes of equilibration and before any infusion, two samples of blood were withdrawn from the tail artery within 10 minutes to document any baseline change in [Mg²⁺]_s (-10 and 0 minutes in Fig 1). After this sampling, animals were infused via the femoral vein catheter with either ISO, PRE, SAL, NPD, or NS vehicle. All agonists were delivered in the same volume of vehicle (0.5 mL), which was infused at a constant rate for 10 minutes. Thus, the total volume of vehicle, the rate of volume infusion, and the duration of infusion were the same for every animal. Agonists for infusion were prepared at concentrations (0.05 µg/mL to 700 µg/mL) that when delivered at the above rate of vehicle infusion, resulted in the desired agonist infusion rate on a microgram per kilogram per minute basis. For experiments requiring pretreatment with ß-adrenergic receptor antagonists, CGP-20712A (2.5 mg/kg IV), atenolol (5 mg/kg IP), butoxamine (10 mg/kg IP), ICI-118551 (0.1 mg/kg IV), PRO (5 mg/kg IP), or an equivalent volume (0.5 mL) of NS were injected either 30 minutes (intraperitoneal drugs) or 15 minutes (intravenous drugs) before the infusion of agonists. The subtype selectivity of the adrenergic agonists and antagonists used in the protocols has been reviewed previously.^{18 19 20} All agonists were administered intravenously to eliminate an absorptive phase, which would delay the onset of the effects of the drugs and potentially complicate data interpretation when agonists were infused in the presence of antagonist. Administration of antagonists, intravenous versus intraperitoneal, was based on previously published literature.^{19 20} The efficacy of all drugs was assessed by the associated hemodynamic effects, which confirmed receptor activation/blockade. The Mg²⁺ present as contaminant in all of the infusion and pretreatment solutions was <1 µmol/L, as measured by AAS (No. 3100, Perkin-Elmer). Each treatment group consisted of six to eight animals. After the infusion, blood samples were withdrawn from the tail artery every 10 minutes. MAP and HR were measured at the time points shown in Fig 1 and at 5 minutes. There were negligible differences in preinfusion MAP or HR between any of the experimental groups. Hct was measured on blood samples from the tail artery of animals in the NS group and in some treated groups. Samples were collected in 75-mm capillary tubes (Fisher Scientific) and centrifuged for 1 minute on a microhematocrit centrifuge (Clay-Adams). The Hct was then read by use of a microcapillary reader (International Equipment Co).

View larger version (18K): [in this window] [in a new window]   Figure 1. Experimental model. Animals were anesthetized with urethane (1 g/kg SC). PE-50 catheters were placed in the tail artery (sampling), femoral vein (infusion), and femoral artery (MAP and HR monitoring). In some groups, adrenergic antagonists were given either 30 minutes (intraperitoneal) or 15 minutes (intravenous) before agonist infusion. After baseline determination of serum Mg2+, agonist or NS was infused for 10 minutes. Serum Mg2+ was then measured at the designated time points. MAP and HR were continuously monitored.

[Mg²⁺]_s Determination
At the designated time points (Fig 1), 0.2 mL of blood, which accounted for the catheter dead space, was withdrawn from the tail artery and discarded. An additional 0.3 mL of arterial blood was removed, and 0.5 mL of NS was infused via the femoral vein catheter to maintain the circulating volume. The whole blood sample was centrifuged (No. 5415, Eppendorf) at 14 000 rpm for 5 minutes in a microfuge tube. Plasma supernatant (25 µL) was diluted in a total volume of 1 mL of 10% (vol) nitric acid and centrifuged (IECCentra-8R, IEC) at 1500 rpm for 10 minutes to precipitate protein. The Mg²⁺ concentration in the resultant serum was measured by AAS. Preinfusion [Mg²⁺]_s was normally distributed (mean, 643 µmol/L; SD, 125; n=122). Since during each protocol Mg²⁺-containing blood was withdrawn and replaced with Mg²⁺-deficient saline to keep the animals euvolemic, small movements of Mg²⁺ into or out of the circulation may go undetected if this "Mg²⁺ dilution" is not acknowledged. Thus, Mg²⁺_c, corrected for changes in hemodilution, was calculated on the basis of [Mg²⁺]_s, the circulating blood volume (BV), and Hct as follows: Mg²⁺_c=[Mg²⁺]_sxBVx(1-Hct). Changes in Mg²⁺_c were then calculated and expressed as micromoles per 300 gbw.

Chemicals
Nitric acid was from Fisher. PRE was from CIBA-GEIGY (Summit, NJ). CGP-20712A was a generous gift from CIBA-GEIGY (Basel, Switzerland). ICI-118551 was from Research Biochemicals International. All the other chemicals were from Sigma Chemical Co.

Statistical Analysis
All data presented are mean±SEM. Data were first analyzed by one-way ANOVA. Multiple means were then compared by Tukey’s multiple comparison test performed with a Mg²⁺ value established for statistical significance of P<.05.

	Results

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ISO Protocol
Fig 2A shows the effects of the infusion of ISO at different rates on [Mg²⁺]_s. Serum Mg²⁺ in NS-infused animals changed by <5% during the entire sampling period. Similarly, little change in serum Mg²⁺ was observed in animals infused with ISO at 0.01 µg · kg^-1 · min^-1. When animals were infused with ISO at 0.1 µg · kg^-1 · min^-1 or higher, [Mg²⁺]_s at 10 minutes was consistently greater than the preinfusion values. The increase in [Mg²⁺]_s continued over time, showing a clear dose-dependent relation at 30 minutes.

View larger version (39K): [in this window] [in a new window]   Figure 2. Time course of changes in [Mg2+]s, HR, and MAP induced by ISO at different rates of infusion. Animals were infused with either NS or ISO at one of the designated infusion rates as described in "ISO Protocol." Data are expressed as mean±SEM. A, Percent change in [Mg2+]s (compared with 0-minute value) vs time. Mg2+ was measured by AAS in the serum of tail artery blood samples and compared with the value at 0 minutes. For clarity, asterisks demonstrating statistical significance have been omitted. At 10 minutes, the change in [Mg2+]s observed only in animals infused with ISO at a rate of 100 µg · kg-1 · min-1 was significantly greater (P<.05) than that of NS-infused control animals. At the latter time points, the changes in [Mg2+]s observed in animals infused with ISO at a rate of 0.1 µg · kg-1 · min-1 or higher were significantly greater (P<.05) than those in NS-infused control animals. B, Change in HR (compared with 0-minute value) vs time. For clarity, asterisks were omitted. At 5 minutes, the increase in HR induced by ISO at 1, 10, and 100 µg · kg-1 · min-1 was significant (P<.05) vs NS. At 10 minutes, the increase induced by ISO at 1 and 10 µg · kg-1 · min-1 was significant (P<.05) vs NS. C, MAP vs time. For clarity, some asterisks were omitted at 5 and 10 minutes. At these time points, MAPs of all ISO-infused groups were significantly different (P<.05) from the NS group. *P<.05 vs NS.

As expected, because the withdrawn blood was replaced with a saline solution, Hct declined over the course of each infusion protocol, but no difference in Hct between the groups at the same time points was detected. Therefore, the Hct values from each group at each time point were combined, and the mean value was used in calculations of Mg²⁺_c. The Hct values at -10, 0, 10, 20, and 30 minutes were 0.48, 0.47, 0.44, 0.44, and 0.42, respectively.

As ISO infusion results in changes in MAP and HR, hemodynamic parameters were monitored to ascertain whether changes in circulating Mg²⁺ correlated with changes in MAP or HR. Infusion of NS resulted in a slight decrease in HR compared with the preinfusion value (Fig 2B). This decrease persisted for the duration of the protocol (-9.0±8.8 bpm at 20 minutes and -7.0±6.1 bpm at 30 minutes). Rats infused with ISO at 0.01 or 0.1 µg · kg^-1 · min^-1 resulted in no significant change in HR versus NS-infused rats. In both these groups, HR declined in the postinfusion period as in the NS group. In contrast, infusion of ISO at a rate of 1.0 µg · kg^-1 · min^-1 or higher resulted in an increase of at least 40 bpm in HR at 5 minutes. A further increase in HR occurred in the animals infused with ISO at 1.0 or 10.0 µg · kg^-1 · min^-1 at 10 minutes, whereas a decline in HR was observed at this time point in animals infused with ISO at 100 µg · kg^-1 · min^-1. At 20 and 30 minutes, HR in animals infused with ISO at 1.0 or 10.0 µg · kg^-1 · min^-1 recovered toward preinfusion levels, whereas the HR remained elevated in animals infused with ISO at 100 µg · kg^-1 · min^-1. The infusion of NS resulted in a small increase in MAP during the infusion period (Fig 2C). After the infusion, MAP in this group decreased from 93.4±4.9 to 83.0±5.0 mm Hg at 20 minutes and to 81.0±4.5 mm Hg at 30 minutes. Infusion of ISO at each rate resulted in a rapid (10 second) fall in MAP to levels lower (P<.05) than NS infusion, which persisted for the duration of the infusion. MAP of animals infused with ISO at rates between 0.01 and 10 µg · kg^-1 · min^-1 recovered to at least 73 mm Hg at 20 minutes and to at least 76 mm Hg at 30 minutes, so that no significant difference between the MAP of control animals and that of animals infused with ISO at these rates was observed at the postinfusion time points. MAP of animals infused with ISO at 100 µg · kg^-1 · min^-1 recovered only partially at the end of the infusion period and remained significantly lower (P<.05) than the MAP of all other groups in this protocol (MAP, 54.0±5.4 mm Hg at 20 minutes and 56.0±6.3 mm Hg at 30 minutes).

The hemodynamic responses of animals in this protocol (an increase in HR and a reduction in MAP) confirm the activation of both ß₁- and ß₂-adrenergic receptors in ISO-treated animals.

To better quantify the changes in Mg²⁺ observed in this protocol, the actual movement of Mg²⁺ into or out of the circulation was calculated, corrected for dilution, and normalized to body weight. These data (the change in Mg²⁺_c, not shown) were qualitatively very similar to those observed in Fig 2A. An increase of 2.88±0.30 µmol Mg²⁺ per 300 gbw occurred over the 30 minutes during and after the infusion of NS. This value was statistically greater (ANOVA, P<.05) than the reference time point of 0 minutes. The data in Fig 2A demonstrate that this small accumulation of magnesium at 30 minutes had a negligible effect on the change in [Mg²⁺]_s. Pretreating the animals with PRO before NS infusion resulted in a modest, but not statistically significant, inhibition (1.73±0.28 µmol Mg²⁺ per 300 gbw) of the elevation seen in the NS control groups. However, after PRO pretreatment, the accumulation of Mg²⁺ was statistically indistinct from the preinfusion reference point. This establishes that some, but not all, of the small rise in Mg²⁺_c observed in control animals was the result of endogenous catecholamine activity. Infusion of ISO at 0.01 µg · kg^-1 · min^-1 resulted in an increase (2.98±0.76 µmol Mg²⁺ per 300 gbw) in the serum Mg²⁺_c that was very similar to that observed in the NS-infused animals. These findings show that despite little change in the measured [Mg²⁺]_s, small but consistent increases in serum Mg²⁺_c were detectable in both control animals and animals infused with ISO at 0.01 µg · kg^-1 · min^-1 when corrections for hemodilution were made. At 30 minutes, Mg²⁺_c in animals infused with ISO at 0.1, 1.0, 10.0, or 100 µg · kg^-1 · min^-1 increased by 7.79±1.10, 8.91±1.10, 9.55±0.77, and 9.63±0.70 µmol Mg²⁺ per 300 gbw, respectively. Each of these changes represented a statistically significant (P<.05) increase in Mg²⁺_c compared with the NS-infused group. The dose-dependent effect of ISO on circulating arterial Mg²⁺ was evaluated by fitting the data at both 10 and 30 minutes to a sigmoid dose-response equation (Fig 3). The group infused with ISO at 100 µg · kg^-1 · min^-1 was excluded from these calculations, since MAP of these animals did not recover after infusion, making them different from the remaining groups. Predicted EC₅₀ values for ISO and maximal responses were obtained for data expressed as both percent change in [Mg²⁺]_s (Fig 3A) and change in Mg²⁺_c (Fig 3B). At 10 minutes, predicted values were as follows: maximal percent increase in [Mg²⁺]_s, 6.4%; EC₅₀ for ISO, 0.02 µg · kg^-1 · min^-1; maximal net increase in Mg²⁺_c, 2.45 µmol/300 gbw; and EC₅₀ for ISO, 0.02 µg · kg^-1 · min^-1. At 30 minutes, predicted values were as follows: maximal percent increase in [Mg²⁺]_s, 19.3%; EC₅₀ for ISO, 0.08 µg · kg^-1 · min^-1; maximal net increase in Mg²⁺_c, 6.7 µmol/300 gbw; and EC₅₀ for ISO, 0.05 µg · kg^-1 · min^-1.

View larger version (22K): [in this window] [in a new window]   Figure 3. Dose-response relation between ISO infusion rate and change in [Mg2+]s. Animals were infused with either NS or ISO at one of the designated infusion rates as described in "ISO Protocol." Mg2+ was measured by AAS in the serum of tail artery blood samples and compared with the value at 0 minutes. Symbols represent data mean±SEM from groups of animals in "ISO Protocol." Lines are predicted values after data were fit to a sigmoid dose-response equation. A, Percent change in [Mg2+]s (compared with 0-minute value) vs infusion rate of ISO. At 10 minutes (T=10 min), the fit equation predicted a maximal response of 6.4% and an EC50 of 0.02 µg · kg-1 · min-1. At 30 minutes (T=30 min), the predicted maximal increase was 19.3%, and the EC50 was 0.08 µg · kg-1 · min-1. B, Change in serum Mg2+ content (compared with 0-minute value) vs infusion rate of ISO. The Mg2+ content of the serum was calculated from the micromolar Mg2+ concentration of a sample circulating blood volume and Hct as described in "Materials and Methods." At T=10 min, a predicted maximal increase of 2.45 µmol Mg2+ per 300 gbw and an EC50 of 0.02 µg · kg-1 · min-1 were obtained. At T=30 min, the predicted maximal response was 6.7 µmol Mg2+ per 300 gbw, and the predicted EC50 was 0.05 µg · kg-1 · min-1.

NPD Protocol
Infusion of ISO resulted in a significant lowering of MAP. To investigate whether the changes in circulating Mg²⁺ observed after ISO infusion were a direct result of the hypotension, a group of animals was infused with NPD to lower MAP through a nonadrenergic mechanism. NPD was infused in this protocol at a rate that mimicked, in duration and intensity, the drop in MAP observed after the infusion of ISO at 1 µg · kg^-1 · min^-1 (MAP, 43.0±2.3 mm Hg at 5 minutes and 45.0±2.0 mm Hg at 10 minutes).

Infusion of NPD resulted in an increase in [Mg²⁺]_s of 7.7±1.7% at 10 minutes, which was slightly higher than that induced by ISO (6.9±1.2% at 10 minutes) (Fig 4A). However, after this initial rise induced by NPD, [Mg²⁺]_s in this group returned to preinfusion levels by 30 minutes and was significantly lower (P<.05) than the values observed in the ISO-infused group at both 20 and 30 minutes. Pretreating the animals with PRO significantly (P<.05) reduced the NPD-mediated increase in [Mg²⁺]_s at 10 minutes by 76% (7.7±1.7% versus 1.8±1.7% increase).

View larger version (32K): [in this window] [in a new window]   Figure 4. Change in [Mg2+]s, HR, and MAP induced by the infusion of NPD in the absence or in the presence of PRO pretreatment. Animals were infused with NPD, in the absence or in the presence of PRO, at rates that mimicked the hypotension induced by infusion of ISO at a rate of 1.0 µg · kg-1 · min-1 as described in "NPD Protocol." Data are expressed as mean±SEM. A, Percent change in [Mg2+]s (compared with 0-minute value) vs time. Mg2+ was measured by AAS in the serum of tail artery blood samples and compared with the value at 0 minutes. *P<.05 vs NPD alone and ISO. **P<.05 vs ISO. B, Change in HR (compared with 0-minute value) vs time. *P<.05 vs ISO. C, MAP vs time.

Infusion of ISO at 1 µg · kg^-1 · min^-1 resulted in an increase in Mg²⁺_c of 3.99±0.40, 5.17±0.50, and 8.90±1.10 µmol Mg²⁺ per 300 gbw at 10, 20, and 30 minutes, respectively (not shown). NPD elevated Mg²⁺_c by 3.90±0.20 µmol/300 gbw at 10 minutes, a change almost identical to that induced by ISO at this time point. However, the Mg²⁺_c of NPD-infused animals decreased at the latest time points, approaching the values observed in NS-treated animals. In spite of identical effects on MAP, significant (P=.05) differences in [Mg²⁺]_s between ISO- and NPD-infused animals were observed at the postinfusion time points. At 30 minutes, the change in Mg²⁺_c following NPD infusion was 2.87±0.50 µmol/300 gbw, a value nearly identical to that reported for the control animals from the previous protocol (2.88±0.30 µmol Mg²⁺ per 300 gbw). Pretreating the animals with PRO significantly reduced (P<.05) the NPD-mediated increase in Mg²⁺_c at 10 minutes, although no significant change in Mg²⁺_c between PRO pretreated and nonpretreated animals infused with NPD was observed at the latter time points.

The hemodynamic profiles of this protocol are shown in Fig 4B and 4C. Infusion of NPD reduced the HR both in the absence and in the presence of PRO pretreatment. NPD alone decreased HR by 31.5±8.4 bpm at 5 minutes and by 29.5±8.6 bpm at 10 minutes. Pretreatment with PRO before infusion of NPD resulted in an decrease in HR of 10.0±5.0 bpm at 5 minutes and 15.0±5.5 bpm at 10 minutes. The decrease in HR with NPD, alone or after pretreatment with PRO, significantly differed (P<.05) from the increase in HR induced by ISO (1.0 µg · kg^-1 · min^-1) at both 5 and 10 minutes. The MAP following NPD infusion in the absence (37.3±1.5 mm Hg at 5 minutes and 42.2±1.7 mm Hg at 10 minutes) or in the presence (42.2±1.7 mm Hg at 5 minutes and 43.2±1.7 mm Hg at 10 minutes) of PRO was nearly identical to that observed in the ISO-infused group.

The data in this protocol demonstrate that a fall in MAP alone is not directly responsible for mobilization of Mg²⁺ into the circulation. PRO had no effect on the NPD-induced fall in MAP but inhibited the NPD-induced increase in Mg²⁺, indicating that the mobilization of Mg²⁺ under these conditions resulted from reflexive ß-adrenergic activity rather than a fall in MAP per se.

Selective Agonist Protocol
To determine whether the ISO-induced increase in Mg²⁺ could be preferentially attributed to either the ß₁- or ß₂-adrenergic receptor subtype, selective agonists were infused. To elicit maximal ß₁- with minimal ß₂-adrenergic activity, PRE, a selective ß₁-adrenergic agonist, was infused at the highest rate possible before the appearance of significant ß₂-mediated effects (hypotension). SAL, a selective ß₂-adrenergic agonist with a longer duration of action than that of ISO, was infused at the lowest rate that resulted in transient blood pressure changes similar to those seen in the ISO-infused animals.

NS minimally changed [Mg²⁺]_s during or after infusion (Fig 5A). The infusion of PRE induced a small decrease in [Mg²⁺]_s, which was not statistically significant when compared with the change in [Mg²⁺]_s observed in the control group. By contrast, infusion of SAL resulted in a significant increase (P<.05 versus NS) in [Mg²⁺]_s at all the postinfusion time points (7.9±1.7%, 12.1±1.6%, and 16.6±2.3% at 10, 20, and 30 minutes, respectively).

View larger version (30K): [in this window] [in a new window]   Figure 5. Change in [Mg2+]s, HR, and MAP induced by the infusion of SAL or PRE. Animals were infused with either NS, SAL, or PRE as described in "Selective Agonist Protocol." Data are expressed as mean±SEM. A, Percent change in [Mg2+]s (compared with 0-minute value) vs time. Mg2+ was measured by AAS in the serum of tail artery blood samples and compared with the value at 0 minutes. *P<.05 vs NS and PRE. B, Change in HR (compared with 0-minute value) vs time. *P<.05 vs NS and SAL. C, MAP vs time. *P<.05 vs NS. **P<.05 vs NS and SAL.

Significant increases (P<.05 versus NS) were also observed in the data presented as the corrected net change in Mg²⁺_c, where infusion of SAL resulted in an increase in the circulating Mg²⁺ content of 3.05±0.26, 4.30±0.52, and 6.50±0.61 µmol per 300 gbw at 10, 20, and 30 minutes, respectively (data not shown). Infusion of PRE elicited a minimal change in Mg²⁺_c at 10 and 20 minutes, whereas an increase of 0.97±0.22, 1.79±0.48, and 2.89±0.50 µmol per 300 gbw at 10, 20, and 30 minutes, respectively, was detected in the NS-infused group, reflecting the previously demonstrated endogenous catecholamine activity. The increase in Mg²⁺_c in the PRE-infused group was lower than that of the NS-treated group, the difference being statistically significant (P<.05) at 20 minutes.

The hemodynamic profiles of this protocol are shown in Fig 5B and 5C. Infusion of NS had little effect on HR during or after the infusion. Also, the infusion of SAL did not significantly change HR. By contrast, infusion of PRE resulted in an increase (P<.05 versus NS and SAL) in HR of 26.0±6.8 bpm at 5 minutes and of 27.5±6.5 bpm at 10 minutes. When compared with the ISO protocol group, this increase in HR was analogous to an infusion of ISO at a rate of between 0.1 and 1.0 µg · kg^-1 · min^-1. Infusion of SAL resulted in a rapid decline in MAP from 85.5±1.9 to 52.3±3.8 mm Hg at 5 minutes and 56.0±4.8 mm Hg at 10 minutes, followed by a rapid recovery. A smaller decline in MAP from 87.5±4.1 to 72.2±5.4 mm Hg at 5 minutes and to 73.8±4.5 mm Hg at 10 minutes was observed in the PRE-infused animals. The decline in MAP in this group was significant (P<.05 versus NS) at 10 minutes. For comparison, in animals infused with ISO at the lowest rate (ie, 0.01 µg · kg^-1 · min^-1), MAP fell from 85.4±3.9 to 60.1±4.7 mm Hg at 5 minutes and to 65.1±5 mm Hg at 10 minutes. In the postinfusion period, MAP of the group infused with PRE recovered to levels indistinguishable from those of the control group.

The hemodynamic profiles demonstrate selective activation of ß₂-adrenergic receptors with SAL and preferential ß₁-adrenergic receptor activation with PRE. The degree of ß₂-mediated hypotension following PRE was significantly less than that seen with ISO at infusion rates that did not alter [Mg²⁺]_s. The change in HR following PRE was equivalent to that seen following infusion of ISO at a rate that resulted in an increase in Mg²⁺.

Antagonist Protocol
To further investigate the role of receptor-subtype specificity in the adrenergic regulation of circulating Mg²⁺, animals were pretreated with either atenolol, CGP-20712A (selective ß₁-adrenergic receptor antagonists), butoxamine, ICI-118551 (selective ß₂-adrenergic receptor antagonists), or PRO (nonselective ß-adrenergic receptor antagonist) before ISO infusion.

Fig 6A shows the changes in [Mg²⁺]_s following the infusion of ISO at a submaximal rate (0.5 µg · kg^-1 · min^-1) either in the absence or the presence of highly selective ß-adrenergic receptor antagonists. Infusion of ISO at this rate resulted in an increase in [Mg²⁺]_s of 9.4±2.3% at 10 minutes, 7.4±2.5% at 20 minutes, and 12.9±1.6% at 30 minutes. These values are in good agreement with those predicted from the previous ISO dose-response curve (Fig 3A). In data not shown, atenolol had no significant effect on the ISO-induced increase in serum Mg²⁺ (8.9±2.2%, 7.9±1.1%, and 7.7±1.9% at 10, 20, and 30 minutes, respectively) despite preventing any ISO-induced increase in HR. Butoxamine (not shown) significantly (P<.05 versus ISO alone) inhibited the ISO-induced increase in serum Mg²⁺ at all postinfusion time points (0.7±1.3%, 2.9±1.5%, and 1.5±2.3%) but had little effect on overall hemodynamic parameters. Fig 6A shows the data from ISO-infused animals pretreated with the highly selective adrenergic antagonists CGP-20712A and ICI-118551 or PRO. Pretreating the animals with CGP-20712A resulted in a marked augmentation of the ISO-induced increase in circulating Mg²⁺ (14.9±3.2%, 17.6±4.3%, and 22.9±3.4% at 10, 20, and 30 minutes, respectively). This effect was statistically significant (P<.05 versus ISO in the absence of pretreatment) at 30 minutes. In contrast, pretreatment with ICI-118551 or PRO almost totally abolished (P<.05) the ISO-induced increase in serum Mg²⁺ at all postinfusion time points (ICI-118551, -0.03±1.7%, 0.5±2.0%, and 4.9±1.2% at 10, 20, and 30 minutes, respectively; PRO, -2.1±3.1%, 0.5±2.8%, and 2.5±0.6% at 10, 20, and 30 minutes, respectively). The data expressed as net change in Mg²⁺_c (not shown) display a qualitatively similar relation.

View larger version (35K): [in this window] [in a new window]   Figure 6. Change in [Mg2+]s, HR, and MAP induced by the infusion of ISO into animals pretreated with CGP-20712A (CGP, a selective ß1-adrenergic receptor antagonist), ICI-118551 (ICI, a selective ß2-adrenergic receptor antagonist), or PRO (a nonselective ß-adrenergic receptor antagonist). Animals were pretreated with either CGP, ICI, or PRO and then infused with ISO as described in "Antagonist Protocol." A, Percent change in [Mg2+]s (compared with 0-minute value) vs time. B, Change in HR (compared with 0-minute value) vs time. C, MAP vs time. Data are expressed as mean±SEM. *P<.05 vs ISO in the absence of any antagonist pretreatment.

The hemodynamic responses of this protocol are shown in Fig 6B and 6C. Infusion of ISO at 0.5 µg · kg^-1 · min^-1 resulted in an increase in HR of 40.0±9.2 bpm at 5 minutes and 42.5±10.6 bpm at 10 minutes. Pretreating the animals with either CGP-20712A (7.5±11 bpm) or PRO (0±8.7 bpm) significantly blocked the ISO-induced increase in HR. The inhibition persisted at 10 minutes in CGP-20712A–pretreated animals (-5±10 bpm), whereas the difference in PRO-pretreated animals was still present but no longer statistically significant. Pretreating the animals with ICI-118551 had no significant effect on the ISO-induced increase in HR. At 20 and 30 minutes, the change in HR was very similar in all groups. Infusion of ISO at a rate of 0.5 µg · kg^-1 · min^-1 resulted in a fall of MAP from 87.8±3.3 to 43.3±2.8 mm Hg at 5 minutes and to 43.7±1.8 mm Hg at 10 minutes. This large drop in MAP was significantly attenuated (P<.05 versus ISO without pretreatment) by pretreating the animals with either ICI-118551 (MAP, 85±2.0 mm Hg at 5 minutes and 84±1.9 mm Hg at 10 minutes) or PRO (MAP, 81±5.6 mm Hg at 5 minutes and 76.5±5.7 mm Hg at 10 minutes). Pretreating the animals with CGP-20712A had little effect on the ISO-induced drop in MAP (MAP, 48.7±3.3 mm Hg at 5 minutes and 46±1.4 mm Hg at 10 minutes).

The hemodynamic profiles here confirmed selective ß₁-adrenergic receptor blockade in the presence of CGP-20712A, selective ß₂-adrenergic receptor blockade in the presence of ICI-118551, and nonselective ß-adrenergic receptor blockade in the presence of PRO.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The data reported in the present study demonstrate that the infusion of the nonselective ß-adrenergic agonist ISO induces a dose-dependent increase in circulating Mg²⁺ in the anesthetized rat. This effect appears to be selectively mediated through activation of the ß₂-adrenergic receptor subtype in that it is reproduced by the infusion of SAL but not PRE. In fact, PRE resulted in an increase of Mg²⁺ in the circulation that was less than even the small amount observed in NS-infused animals. The ISO-induced increase in circulating Mg²⁺ was inhibited in the presence of a highly selective ß₂-adrenergic receptor antagonist or a nonselective ß-adrenergic receptor antagonist. In the presence of a highly selective ß₁-adrenergic receptor antagonist, the ISO-induced increase in circulating Mg²⁺ was amplified, supporting the concept of differential regulation of circulating Mg²⁺ through ß₁- and ß₂-adrenergic receptor subtypes and suggesting that ß₁-adrenergic stimulation might mediate a loss of Mg²⁺ from the circulation.

The NPD protocol provided important data demonstrating that our observations cannot simply be explained as a nonrelevant effect of large doses of drug or as being secondary to tissue ischemia. The increase in circulating Mg²⁺ observed in this protocol, which provided a more (patho)physiological model of adrenergic stimulation, induced a significant increase in Mg²⁺, which was largely inhibited by PRO, indicating that the process was reflexively mediated through ß-adrenergic mechanisms and not due to hypotension/tissue ischemia per se.

Time Course
The effects of ß-adrenergic stimulation on circulating Mg²⁺ and cardiovascular parameters (HR and MAP) were manifested over very different time courses. In fact, serum Mg²⁺ continued to increase for up to 20 minutes after the infusion of ISO or SAL was discontinued, at which point the cardiovascular effects of the agonists had dissipated. Since it has been reported that ISO is present in the circulation up to 1 hour after its infusion into rats,²¹ it is likely that the ISO infused in our protocol would persist in the circulation at significantly high levels for the duration of the protocols. However, this would not explain the discrepancy between the resolution of the cardiovascular effects and the persistent increases in circulating Mg²⁺. The data from the NPD protocol demonstrate that in response to reflexive adrenergic stimulation, the elevation in circulating Mg²⁺ is transient in nature. The possibility exists that the process responsible for the increase in Mg²⁺ does not exhibit desensitization and/or downregulation of the adrenergic receptor. It has been reported that the infusion of ISO results in the release of several other agents, eg, glucagon,²² which can induce an increase in circulating Mg²⁺ in the rat.²³ Thus, some or all of the Mg²⁺ increase we observed may have been indirectly induced by ISO through this or other secondary agents, resulting in a delay in the maximal response. When our data were analyzed and the predicted values for EC₅₀ were obtained, we found a threefold to fourfold difference in the values at 10 and 30 minutes. This suggests that a two-component process may be occurring, an early effect directly mediated by ISO and a latter effect possibly mediated by additional effectors.

ß-Adrenergic Subtypes
The observation that an increase in circulating Mg²⁺ in response to ISO is mediated via stimulation of ß₂- but not ß₁-adrenergic receptors is interesting in that cAMP is the principal second messenger for both of these ß-adrenergic receptor subtypes.¹⁸ Although the present study provides clear evidence that circulating Mg²⁺ is elevated in response to stimulation of one receptor subtype but not the other, our data do not provide a mechanistic explanation for the differential effect; thus, we can only speculate as to how this occurs. One obvious explanation would be that the effects that we observed were simply due to the fact that in the rat, it is generally assumed that there are more ß₂- than ß₁-adrenergic receptors present in the whole body. Although this is one simplistic explanation for our results, we were unable to find, in the current literature, a generally accepted ratio of ß₂- to ß₁-adrenergic receptors in the whole rat. One could derive this number from studies done on individual tissues; however, this method is complicated by variability between values based on the age and sex of the animal and other factors that may alter the ratio. Elements other than receptor number may also be responsible for the effects we observed. It has been shown that the ß₂-adrenergic receptor couples to adenylate cyclase more efficiently than does the ß₁-adrenergic receptor subtype. And although this observation of differential coupling of the ß-adrenergic receptor to adenylate cyclase has been characterized mostly in hearts,^{24 25 26 27} transfection studies demonstrate²⁸ that the phenomenon is intrinsic to the receptor itself and therefore more likely to be present in ß-adrenergic receptors in all tissues. Additionally, there have been reports suggesting non–cAMP-mediated signaling initiated by stimulation of the ß₂-adrenergic receptor.^{29 30 31}

The possible role of atypical (ß₃) adrenergic receptors in this model is also only speculative. It is of interest to note that ß₃-adrenergic receptors do not appear to desensitize or downregulate as the other adrenergic receptor subtypes³² do and that the EC₅₀ of this receptor for ISO is approximately fivefold higher than that of the ß₂-adrenergic receptor.³³ These characteristics may help to explain both the persistent effect observed in the present data and the different EC₅₀ values obtained at 10 and 30 minutes.

Previous Literature
The present study is the latest of many that have attempted to characterize Mg²⁺ homeostasis in vivo after adrenergic stimulation.^{34 35 36 37 38 39 40 41 42 43 44 45} The data available in the current literature have been obtained by using experimental models with some important differences that need to be considered when attempting to make direct comparisons. The majority of previous work in this field has been performed by using nonanesthetized humans infused with epinephrine at relatively low rates over time periods significantly longer than ours and has consistently shown a decrease in serum Mg²⁺ over time.^{35 36 37 38 39 40 41 42 43} Infusing SAL into unanesthetized humans at a rate of 0.12 µg · kg^-1 · min^-1 for 60 minutes resulted in a significant decrease in serum Mg²⁺ after 30 minutes³⁶ with a nadir of 10% below preinfusion levels occurring 60 minutes after the end of the infusion. Infusion of albuterol (also a selective ß₂-adrenergic agonist) at 67 ng per second for 240 minutes produced no change in serum Mg²⁺ at the end of the infusion period but resulted in an increase in the urinary excretion of Mg²⁺.⁴³ ISO infused into unanesthetized human subjects at a rate that increased HR by 20% did not change circulating Mg²⁺.⁴² Direct comparison of adrenergic responses between species is problematic, since in the cardiovascular system,⁴⁶ for example, the relative proportions of ß-adrenergic subtypes differ between species. The use of different adrenergic agonists also complicates comparisons. Potency hierarchies of the various ß-adrenergic agonists at the individual ß-adrenergic receptor subtypes have been established.^{47 48} At the ß₁-adrenergic receptor, the potency rank order is ISO>norepinephrineepinephrine, and at the ß₂-adrenergic receptor, the order is ISO>epinephrine>norepinephrine. Early reports suggest that the order at the ß₃-adrenergic receptor is norepinephrine>ISO>epinephrine. Thus, our protocol using ISO provides a more potent nonspecific stimulation of ß-adrenergic receptors compared with protocols that use epinephrine or norepinephrine. Additional features regarding agonist selection that require scrutiny are the rate and duration of infusion. Dosages of drugs are better compared between species in reference to body surface area rather than to body mass.⁴⁹ A drug administered at the same microgram per kilogram dose to a rat and a human will result in the human receiving a dose approximately five times larger than the rat on the basis of surface area.

There have been two prior studies that have used an experimental model similar to ours. One investigator⁴⁴ examined the effect of adrenergic stimulation on circulating Mg²⁺ by infusing ISO into rats at a rate of 0.2 µg/min (average weight of animals, 0.32 kg; equivalent rate per kilogram, 0.625 µg · kg^-1 · min^-1) for 40 minutes and found no change in serum Mg²⁺ level, although no corrections were made for hemodilution in this study. Another group of animals was infused with epinephrine, either in the absence or in the presence of either an - or ß-adrenergic blocker. A small but statistically significant increase in Mg²⁺ following infusion of epinephrine in the presence of -adrenergic blockade was observed. This increase was attributed to hemodynamic instability, although the systolic blood pressure was never below 93 mm Hg in this group. It was subsequently shown⁴⁵ that ISO, injected subcutaneously into rats, induced a dose-dependent increase in plasma Mg²⁺ that reached a maximum (doubling of Mg²⁺) 2 hours after injection.

Thus, the present study is consistent with the findings of previous investigators using experimental models resembling ours in which ß-adrenergic stimulation with ISO, or epinephrine in the presence of -blockade, induced elevation of circulating Mg²⁺.^{44 45} Our data eliminate the ß-adrenergic–induced hemodynamic changes as being the proximate cause of Mg²⁺ mobilization. In addition, we describe a dose-response relation with a calculated EC₅₀ and characterize a time course for the response.

The Source of Mg²⁺
It should be emphasized that despite the ability to consistently detect dose-dependent increases in serum Mg²⁺ in response to ß-adrenergic stimulation, our preliminary work has not revealed a measurable net gain or loss of Mg²⁺ in the tissues that we have studied, ie, heart, liver, kidney, skeletal muscle, lung, and erythrocytes. We measured hemodynamic parameters only to ascertain the effectiveness of receptor activation/blockade and do not mean to imply that we believe the cardiovascular system to be the source of the Mg²⁺. One possibility is that the Mg²⁺ increase in the serum is derived from multiple sources. The actual change in any single tissue Mg²⁺ would then be of a magnitude that was less than the error of our detection system; thus, these changes were not measurable. For example, we measured the total organ Mg²⁺ content of the liver to be 162 µmol. If the liver were the exclusive source of Mg²⁺ in our model, the loss in that tissue would be small compared with the total Mg²⁺ content of the tissue. If more than one tissue were the source, then the change in any one tissue, relative to total Mg²⁺ content, would be even smaller. We measured the total Mg²⁺ content of the heart to be 9.2 µmol. The heart would need to be almost totally depleted of Mg²⁺ if it were the exclusive source of Mg²⁺ responsible for the increase observed in the serum. It is more likely then that multiple tissues were responsible for the changes in serum Mg²⁺ that we observed. We are presently addressing the issue of individual tissue Mg²⁺ homeostasis with isolated/perfused organ studies, which will allow more precise data via arteriovenous differences. Another consideration is that changes in circulating Mg²⁺, including blood within the parenchyma of organs themselves, artifactually conceal measurable changes in tissue homogenates. Alternatively, Mg²⁺ may have been mobilized from tissue(s) that we did not evaluate, such as bone or brain. The involvement of the kidney, through filtration/reabsorption, was briefly considered. However, on the basis of the preinfusion [Mg²⁺]_s (642 µmol/L) and reported values for the proportion of serum Mg²⁺ that is filtered (80%⁵⁰ ), the glomerular filtration rate (1.62 mL/min⁵¹ ), and the fractional excretion (17%⁵¹ ) of Mg²⁺, it is not likely that this mechanism contributed significantly to our observations. In response to the improbable event of the fractional excretion of Mg²⁺ decreasing to zero immediately as drug infusion is started, serum Mg²⁺ content would be predicted to increase by 1.4 µmol every 10 minutes. At 10 minutes, this would have accounted for only 35% of the increase in Mg²⁺ content observed in the NPD-infused animals. At 30 minutes, less that half of the increase in Mg²⁺ content observed in the animals infused with ISO at a rate of 10 µg · kg^-1 · min^-1 would be accounted for by these mechanisms.

Significance of an Increase in Circulating Mg²⁺
The present findings raise the important question as to the significance of the increase in circulating Mg²⁺ in response to ß₂-adrenergic stimulation. Assuming that in our model Mg²⁺ was mobilized from intracellular stores to the circulation and that the amount of cation mobilized was of a sufficient magnitude to modify the concentration of free Mg²⁺, we can infer that the various Mg²⁺-sensitive processes at these intracellular sites would be altered. In the liver, it has been shown that relatively small changes in total Mg²⁺ content directly effect the concentration of free Mg²⁺.⁵²

The increase in Mg²⁺ in the blood may itself be a significant physiological response. Infusion of Mg²⁺ into various mammals has demonstrated that several important hemodynamic parameters are directly affected by an increase in the level of circulating Mg²⁺.^{53 54 55} James et al,⁵⁶ using baboons as subjects, studied the effects of intravenous Mg²⁺ bolus doses on hemodynamic parameters during infusion of adrenaline. Infusion of Mg²⁺, at a dose that raised the serum concentration fivefold, significantly reduced the adrenaline-induced increases in systemic vascular resistance, systolic blood pressure, and diastolic blood pressure and abolished the arrhythmias associated with adrenaline infusion. From studies performed in vitro, it has been suggested that Mg²⁺ can regulate catecholamine release from both peripheral and adrenal sources.^{57 58} Thus, it is possible that the increase in circulating Mg²⁺ that we observed in response to ISO may constitute an endogenous modulator of catecholamine activity. DiPette et al⁵⁹ infused Mg²⁺ into normal rats at a dose that raised [Mg²⁺]_s by a factor of 1.5. The authors noted no systemic hemodynamic consequences of the infusion but did observe an increase in flow in the coronary circulation. Thus, elevated Mg²⁺ in the circulation in response to adrenergic stimulation may augment blood flow, and thus O₂ delivery, to the myocardium at a time when the demand is expected to increase. Alternatively, the increase in circulating Mg²⁺ may be an epiphenomenon of no physiological significance other than being a reservoir for the Mg²⁺ extruded from intracellular compartments. In either case, the increase in circulating Mg²⁺ results in a condition in which total body Mg²⁺ homeostasis is modified by increased renal excretion of Mg²⁺.^{50 60}

Conclusions
The present study adds to and strengthens our in vitro results, which have shown ß-adrenergic stimulation to be an important regulator of Mg²⁺ homeostasis. The NPD protocol demonstrates that reflexive adrenergic stimulation following hypotension provides sufficient adrenergic activity to induce the elevation in circulating Mg²⁺, raising the possibility that the translocation of Mg²⁺ into the circulation may be an important component or consequence of the stress response. We have also demonstrated that the effect is mediated differentially through the ß-receptor types, whereby stimulation of the ß₂-adrenergic receptor, but not the ß₁-adrenergic receptor, induces an increase in circulating Mg²⁺. Our data suggest that the ß₁-adrenergic receptor might promote a loss of Mg²⁺ from the circulation. The significance of our work, as well as that of others, will hopefully become more clear in the future as the actual mechanisms of Mg²⁺ membrane transport are further elucidated and specific inhibitors of these mechanisms become available.

	Selected Abbreviations and Acronyms

 

AAS = atomic absorbance spectrophometry gbw = grams body weight Hct = hematocrit HR = heart rate ISO = isoproterenol MAP = mean arterial pressure Mg2+c = corrected serum Mg2+ content (µmol/300 gbw) [Mg2+]s = serum Mg2+ concentration (µmol/L) NPD = nitroprusside NS = 0.9% saline PRE = prenalterol PRO = propranolol SAL = salbutamol

	Acknowledgments

 
This study was supported by National Institutes of Health grant HL-18708 and the Allen Scholar Research Fund, Department of Surgery, Case Western Reserve University.

	Footnotes

 
Reprint requests to Dr A. Scarpa, Department of Physiology and Biophysics, Case Western Reserve University, School of Medicine, 2109 Abington Rd, Cleveland, OH 44106. E-mail axs15@po.cwru.edu.

Received December 23, 1994; accepted July 31, 1995.

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