(Hypertension. 1997;29:1173-1177.)
© 1997 American Heart Association, Inc.
Articles |
Metformin Inhibits Ganglionic Neurotransmission in Renal Nerves
From the Department of Pharmacology, The Panum Institute, University of Copenhagen, Denmark (J.S.P.), and Department of Pharmacology, Louisiana State University Medical Center, New Orleans.
Correspondence to Jørgen Søberg Petersen, MD, PhD, Department of Pharmacology, The Panum Institute, Bldg 18.6, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark. E-mail fijsp{at}farmakol.ku.dk
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Abstract |
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Abstract Intravenous administration of the antihyperglycemic agent metformin decreases arterial pressure and sympathetic nerve activity (SNA). To test the hypothesis that metformin inhibits SNA by interrupting ganglionic neurotransmission, we compared the actions of intravenous administration of metformin and the ganglionic blocker trimethaphan on postganglionic renal and preganglionic adrenal sympathetic nerves in pentobarbital-anesthetized male Sprague-Dawley rats. Intravenous metformin elicited dose-dependent decreases in postganglionic renal SNA (1 mg/kg: 0±0%; 10 mg/kg: -20±4%; 100 mg/kg: -92±3%; n=7). Conversely, only the maximal dose of metformin affected preganglionic adrenal SNA (100 mg/kg:
adrenal
SNA=-14±6%; n=8). Ganglionic blockade with intravenous
trimethaphan (5 mg/kg) produced a differential
sympathoinhibitory response similar to the response
observed after high-dose metformin (renal SNA=-100±3%; adrenal
SNA=-17±7%; P<.001). Preganglionic renal neurons were
electrically stimulated in the spinal cord, before and during the peak
of the sympathoinhibitory response to
intravenous metformin, and the magnitude of the
stimulus-evoked increases in postganglionic renal SNA were compared.
Metformin dose-dependently attenuated the magnitude of the increase in
postganglionic renal SNA elicited by stimulation of the spinal cord (30
mg/kg: -23±8%; 90 mg/kg: -65±11%; 270 mg/kg: -91±8%; n=6 per
dose). We conclude that high-dose intravenous metformin
interrupts ganglionic neurotransmission in renal nerves.
Key Words: metformin • sympathetic nerve activity • neurotransmission • diabetes mellitus
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Introduction |
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Metformin is an antihyperglycemic agent that has been widely used in Europe, Australia, and Canada for almost 40 years and was recently approved in the United States for the treatment of type 2 diabetes.1 2 3 Whereas metformin consistently improves insulin sensitivity in type 2 diabetics and decreases plasma lipids in hyperlipidemic patients, the effects of metformin on arterial blood pressure are conflicting. Metformin does not affect blood pressure in normotensive or mildly hypertensive patients; however, an antihypertensive action has been reported in obese, hypertensive, type 2 diabetic patients, who are characterized by elevated sympathetic nerve activity.4 5 Petersen and DiBona6 demonstrated that acute intravenous administration of metformin elicits marked renal sympathoinhibitory and hypotensive responses in anesthetized spontaneously hypertensive rats (SHR) that are not mediated through stimulation of the vagus nerve or release of nitric oxide or prostaglandins. However, the sympathoinhibitory and hypotensive responses to intravenous metformin are similar in intact and baroreceptor-denervated rats,6 suggesting that intravenous metformin inhibits the sympathoexcitatory reflex response that would be expected during metformin-induced hypotension.
Muntzel and Petersen7 demonstrated that the acute
hypotensive response to intravenous metformin is blocked by
either ganglionic blockade or 
-adrenergic blockade, suggesting that
the metformin-induced decrease in arterial pressure is
meditated by generalized withdrawal of sympathetic tone.7
Overall, these findings are compatible with a ganglionic blocking
action of intravenous metformin, and therefore, the purpose
of this study was to test the hypothesis that metformin decreases
sympathetic nerve activity by blocking ganglionic neurotransmission in
sympathetic nerves. To test this hypothesis, we compared (1) the
changes in postganglionic renal sympathetic nerve activity (RSNA) and
preganglionic adrenal sympathetic nerve activity (ASNA) elicited by the
intravenous administration of metformin, and (2) the
magnitude of the increases in postganglionic RSNA elicited by
electrical stimulation of the thoracic spinal cord (T10 through T12) in
the region of the intermediolateral nucleus before and after
intravenous administration of metformin. This region of the
spinal cord contains the cell bodies of the preganglionic sympathetic
neurons providing input to the renal sympathetic
nerves.8 9 The short-acting ganglionic blocking agent
trimethaphan was used as a reference drug.
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Methods |
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Animal Preparation
Experiments were performed in male Sprague-Dawley rats (275 to 325 g, Harlan, Indianapolis, Ind). All procedures were in accordance with National Institutes of Health guidelines for the care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee at Louisiana State University Medical Center. Rats were anesthetized with pentobarbital (60 mg/kg IP) and placed on a water-filled heating pad maintaining rectal temperature at 37°C to 38°C. Polyethylene catheters (PE-10 fused to PE-50) were inserted into a femoral artery and vein for measurement of mean arterial pressure (MAP) and intravenous infusion of isotonic saline (3.0 mL/h) and pentobarbital (10 mg/kg per hour), respectively. An additional catheter was inserted into the right jugular vein for intravenous administration of drugs. Arterial pressure was measured with a pressure transducer (Statham P23XL) and displayed continuously on a polygraph (Grass model 7D). Heart rate was recorded by a linear cardiotachometer (Grass model 7P4) triggered by the arterial pressure waveform. An endotracheal tube was inserted and the rat mechanically ventilated with room air. During stimulation experiments, the rats were paralyzed with pancuronium (1.0 mg/kg IV bolus, followed by 1.0 mg/kg per hour) after the continuous pentobarbital infusion was started.
The left adrenal or renal sympathetic nerve was isolated and placed on a bipolar platinum electrode. The nerve activity was led through a high-impedance probe (Grass model HIP511) and amplified (3000 to 20 000 times) and filtered (30 to 3000 Hz) with a band-pass amplifier (Grass model P511). The amplified and filtered signal was led to an oscilloscope (Tektronix 5113) and an audio amplifier/loudspeaker (Grass model AM8). The nerve signal was converted to slow wave activity with a moving averager (CWE model M821, 100-millisecond time constant) and integrated with a rectifying voltage integrator (Grass model 7P10) and displayed on the chart recorder. The recording electrode was fixed to the nerve with dental impression material (President Light Body, Coltène AG). To eliminate afferent nerve activity from the recorded signal, nerve bundles were crushed distal to the electrodes. The level of background noise was determined after the rat was killed (25 mg pentobarbital IV).
For those studies in which the spinal cord was electrically stimulated, anesthetized rats were placed in a stereotaxic headholder and spinal investigation unit (Kopf Instruments). Vascular catheters and a renal nerve recording electrode were placed as described above, and the spinal cord was exposed by bilateral full laminectomy from T10 to L1.
Experimental Protocol
RSNA Versus ASNA Responses to Intravenous Metformin
To examine the effect of intravenous metformin on
postganglionic RSNA and preganglionic ASNA, we administered metformin
(1, 10, and 100 mg/kg IV) to groups of rats prepared for
recording of either RSNA (n=7) or ASNA (n=8). Metformin was
administered in cumulative doses, with 15 minutes of recovery allowed
between each dose.6 Injection volume was 1 mL/kg at all
doses. To evaluate the homogeneity of preganglionic and postganglionic
sympathetic nerve fibers in the renal and adrenal nerves, an
intravenous injection of a maximal dose of the short-acting
ganglionic blocker trimethaphan (5 mg/kg) was given after rats had
recovered from the last dose of metformin.
Effects of Intravenous Metformin on Evoked
Postganglionic Potentials Elicited by Stimulation of the Spinal
Intermediolateral Nucleus
The tip of a concentric bipolar stimulating electrode (NE-100,
Rhodes Medical Instruments) was placed 0.5 mm lateral to the
sulcus medianus posterior and 0.25 mm ventral of the dorsal
arachnoidea spinalis between T10 and T12. A stimulator (Grass S8800)
and constant current unit were used to deliver cathodal square-wave
pulses (0.5 to 1.0 mA, 1-millisecond duration, 0.5 Hz). Evoked
potentials in the postganglionic renal nerve were averaged (40 to 50
sweeps) with an RC Electronics Computer Scope System. While
stimulating, the electrode was moved ventrally through the spinal cord
in 200-µm steps until the site producing the largest potential was
identified and the response threshold determined. When required,
additional tracks were made in the same rostral-caudal plane. The
electrode tracks were separated by 500 µm. For each experiment,
the site having the lowest threshold and longest onset of latency was
used. For drug testing, a stimulus current two to three times threshold
was used. Potentials evoked in the postganglionic renal nerve were
measured before and during maximal metformin-induced suppression of
RSNA as well as after the recovery of RSNA to control levels. The
intravenous doses of metformin used were 30, 90, and 270
mg/kg (n=6 per dose).
Drugs
Pentobarbital sodium (50 mg/mL; Nembutal, Abbott Laboratories)
was diluted in isotonic saline. Pancuronium (1 mg/mL; Astra
Pharmaceutical Products, Inc) was dissolved in isotonic saline and
stored at room temperature. Trimethaphan camsylate (Arfonad, Roche
Laboratories) was diluted in isotonic saline and stored at 5°C.
Metformin (1,1-dimethylbiguanide; Sigma Chemical Co) was dissolved in
isotonic saline and prepared fresh before each experiment.
Statistics
Overall statistical analysis of one-way–classified data
(treatment group) was performed with one-way ANOVA. Overall statistical
analysis of two-way–classified data (treatment group and dose)
was performed by repeated measures ANOVA. Student's paired or unpaired
t test with Bonferroni correction for multiple comparisons
was used for comparisons of one-way classified data within or between
groups.10 Differences were considered significant at a
value of P<.05. All values are mean±SE.
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Results |
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Fig 1
shows representative tracings
from two rats in which either RSNA (left) or ASNA (right) responses to
metformin and trimethaphan were recorded. Metformin (100 mg/kg IV)
produced an immediate hypotensive and renal
sympathoinhibitory effect similar to the response to
trimethaphan (5 mg/kg IV). Group data for responses to metformin are
summarized in Fig 2. Metformin produced similar
decreases in MAP in RSNA (n=7) and ASNA (n=8) groups; however, the
decreases in RSNA were significantly larger than those elicited in
ASNA. Metformin (100 mg/kg IV) decreased RSNA by 92±3%, whereas ASNA
was only decreased by 14±6% (P<.001). Similarly, the
ganglionic blocker trimethaphan (5 mg/kg) decreased RSNA by 100±3%,
whereas ASNA was decreased by only 17±7% (P<.001).
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Fig 3 shows data from a representative
experiment illustrating the relationship between arterial
pressure, RSNA, and the increase in postganglionic RSNA evoked by
stimulation of the spinal cord in the region of the intermediolateral
nucleus. The average threshold of the evoked potentials was 79±21
µA, and the mean onset of latency from the stimulus to the peak of
the evoked increase in RSNA was 89±8 milliseconds.
Intravenous metformin produced a profound and reversible
decrease in the amplitude of the evoked potential that was greatest
during the peak of the hypotensive and sympathoinhibitory
responses. Metformin (30, 90, and 270 mg/kg) dose-dependently decreased
the magnitude of the evoked increase in RSNA elicited by spinal
stimulation (Fig 4).
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Discussion |
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Previous studies have shown that intravenous metformin produces a sympathoinhibitory response that overrides the ability of the arterial baroreflex to buffer a decrease in arterial pressure with a reflex-mediated increase in peripheral sympathetic nerve activity.6 Moreover, the hypotensive response to intravenous metformin is blocked by
-adrenoceptor
blockade or ganglionic blockade, suggesting that it is mediated by a
generalized decrease in sympathetic nerve activity.7 In
accordance with these previous observations, the present results
indicate that the sympathoinhibitory response elicited by
intravenous metformin results from the action of this drug
to interrupt neurotransmission at sympathetic ganglia. Two lines of
evidence support this conclusion. First, intravenous
metformin dose-dependently decreased postganglionic RSNA but not
preganglionic ASNA. The postganglionic and preganglionic content of
these two nerves was verified with the nondepolarizing, nicotinic
receptor antagonist trimethaphan.11 Ganglionic
blockade with trimethaphan elicited decreases in RSNA and ASNA similar
to those elicited by the highest dose of metformin. Stimulation of cell
bodies of the preganglionic sympathetic neurons, which provide input to
the postganglionic renal nerves, resulted in an evoked increase in
postganglionic RSNA. The ability of metformin to decrease the amplitude
of the evoked increase in postganglionic RSNA reflects the ability of
the drug to prevent transmission of the preganglionic impulses to the
postganglionic fibers at the level of the sympathetic ganglia. It could be argued that metformin decreased postganglionic RSNA via a nonselective, local anesthetic action on peripheral autonomic nerves or had an effect on the spinal preganglionic neurons in the intermediolateral nucleus. However, the fact that intravenous metformin did not decrease ASNA argues against both of these possibilities.
Petersen and DiBona6 reported that intracerebroventricular administration of metformin also decreases MAP and RSNA in SHR, although the time course of these responses is much longer than that elicited by intravenous administration. The mechanism or mechanisms mediating the central responses are not known, but we speculate that metformin may interrupt neural synaptic transmission by the same mechanism in the central nervous system as in peripheral sympathetic nerves.
Although several studies have demonstrated an antihypertensive action of chronic metformin treatment in humans,4 5 12 13 14 15 the issue of an antihypertensive effect of metformin in humans is still controversial, as other clinical studies have failed to demonstrate an antihypertensive effect.16 17 18 19 20 The reason for these conflicting results is unclear; however, on the basis of our finding that metformin has sympathoinhibitory properties, we have suggested that the antihypertensive action of metformin is related to the level of sympathetic nerve activity before treatment.6 This is consistent with the lack of a hypotensive action in normotensive humans.16 18 20 In the SHR, chronic oral metformin treatment inhibits the sympathoexcitatory response to hyperinsulinemia and prevents the development of hypertension,21 22 23 whereas chronic metformin treatment does not affect blood pressure in normotensive Wistar rats; Dahl salt-sensitive rats; or one-kidney, one clip hypertensive Sprague-Dawley rats.22 24 These findings concur with the fact that sympathetic nerve activity is known to be elevated in the SHR relative to most other rat models.25 Thus, although metformin may decrease arterial pressure in hypertensive individuals by other mechanisms as well,26 27 the sympathoinhibitory action of metformin may be an important therapeutic effect in hypertensive states with elevated sympathetic tone (eg, the obese, hypertensive, type 2 diabetic individual4 5 ).
In conclusion, intravenous administration of metformin inhibits ganglionic neurotransmission in renal nerves in anesthetized normotensive rats. We ascribe the marked, acute hypotensive response to intravenous metformin to generalized inhibition of ganglionic neurotransmission.
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Acknowledgments |
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This work was performed during the tenure of J.S.P. as a Visiting Assistant Professor at the Department of Pharmacology and Experimental Therapeutics, Louisiana State University Medical Center, New Orleans. This study was supported by grants from the Danish Research Council for Health Sciences, the Danish Diabetes Association, Fonden til Lægevidenskabens Fremme, Eva and Robert Voss Hansen Foundation, and the Ruth Kønig-Petersen Foundation. K.J.V. and D.R.K. were supported by grants from the National Institute on Drug Abuse (DA 08255) and the National Institute of Diabetes and Digestive and Kidney Diseases (DK 43337).
Received October 8, 1996; first decision November 4, 1996; accepted December 6, 1996.
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