- Email: [email protected]
A possible indirect sympathomimetic action of metformin in the arterial vessel wall of spontanously hypertensive rats
Life Sciences 69 (2001) 1085–1092
A possible indirect sympathomimetic action of metformin in the arterial vessel wall of spontanously hypertensive rats JiHun M. Lee, Jacob D. Peuler* Department of Pharmacology, Midwestern University, 555 31st Street, Downers Grove, IL 60515, USA Received 7 August 2000; accepted 30 January 2001
Abstract The antidiabetic drug metformin (MF) typically achieves only micromolar levels in plasma with normal therapeutic use. However, it is also known to accumulate in various tissues up to several times higher after standard oral dosing and we now have evidence from both in vivo and in vitro experiments with spontaneously hypertensive rats (SHR) that millimolar levels stimulate release of norepinephrine (NE) from vascular sympathetic nerve endings (SNEs). As shown in the present work with SHR tail arterial tissue (rich in SNEs), the known vasodilator effect of millimolar levels of MF on the smooth muscle (even if contracted with a nonadrenergic agonist), is attenuated by the presence of the SNEs unless phentolamine (an alpha receptor blocker) is present. We reasoned that the mechanism for this apparent NE-releasing action of MF is not exocytotic release as that would require depolarization of the neuronal cell membranes in SNEs, and MF at millimolar levels is known to repolarize (not depolarize) membranes of other cells. Thus, we tested the possibility that MF releases NE by an indirect sympathomimetic-like action. Such an action should be amplified by monoamine oxidase inhibitors (e.g. iproniazid) and blocked by NE-carrier inhibitors (e.g. desipramine). Accordingly, we found that the abovementioned attenuating effect of intact SNEs on MF’s relaxation of SHR tail arterial tissue (compared to tissues in which SNEs were experimentally removed with 6-hydroxydopamine) was amplified nearly 3-fold by iproniazid (p,0.05) and blocked by desipramine (p,0.05). These results support an indirect sympathomimetic action of MF and raise the question whether commonly used antidepressants with properties similar to iproniazid and desipramine might alter MF’s beneficial vasodilatory (and thus antihypertensive) effectiveness in diabetic patients with hypertension. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Norepinephrine; Adrenergic nerve endings; Vasorelaxation; Diabetes; Antidepressants
* Corresponding author. Tel.: (630) 515-6068; fax: (630) 971-6414. E-mail address: [email protected] (J.D. Peuler) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 2 0 2 -4
1086
J.M. Lee, J.D. Peuler / Life Sciences 69 (2001) 1085–1092
Introduction We have found that the antidiabetic drug metformin at concentrations above 0.1 mmol/L rapidly relaxes adrenergic contractions in vitro in intact vascular tissue rings prepared from the ventral tail artery of the female spontaneously hypertensive rat (SHR) [1]. This acute relaxant effect is not likely limited to just the tail artery as we have also found that bolus intravenous (iv) injection of metformin (at doses likely to produce such concentrations in plasma) rapidly decreases not only resting arterial pressures in such SHR but also pressures supported specifically by iv infusion of exogenous a-adrenergic agonists [2]. However, these acute depressor responses to iv metformin in these same SHR were accompanied by immediate increases in arterial plasma norepinephrine (NE) levels; increases not dependent on the presence of the adrenomedulla and seen in ganglionically-blocked as well as autonomically-intact animals [2]. Thus, we know that they are neither adrenomedullary in origin nor reflexly-mediated. Rather, we concluded from that in vivo work that metformin can directly stimulate release of NE from postganglionic sympathetic nerve endings independent of axonal action potentials [2]. However, we were not able to determine in an in vivo study of that type whether such a neural action of metformin was at all functionally important in the arterial vessel wall. For example, does it compromise the direct relaxant action of the drug on the underlying smooth muscle? The ventral tail artery of the rat is rich in sympathetic nerve endings as well as smooth muscle cells [3,4]. Thus we decided to re-examine the abovementioned acute relaxant effect of metformin on adrenergic contractions in vitro in tail arterial tissue isolated from female SHR but this time after its pretreatment with 6-hydroxydopamine (6-OHDA) which specifically removes sympathetic nerve endings without altering the ability of the underlying smooth muscle to either contract or relax [3,5]. We hypothesized that this pretreatment would notably enhance metformin’s relaxant effect or, conversely, that the presence of intact sympathetic nerve endings would notably attenuate its relaxant effect. Indeed, such results were observed and thus we conducted additional experiments in an attempt to ascertain the mechanism responsible for this effect. We focused on the possibility that metformin may be releasing NE from nerve endings by way of an indirect sympathomimetic-like action. Thus, as expected with such an action, we hypothesized that a monoamine oxidase (MAO) inhibitor would enhance it (i.e. enhance the attenuating effect of the nerve endings on metformin’s relaxation) and a NE-carrier inhibitor would abolish it. Methods As in the previous work that led up to this study [2], we used adult female SHR in the present experiments. They were housed as described previously under conditions known to desynchronize estrous cycles [2]. Then they were selected at random (one rat per day) and anesthetized with ketamine/xylazine (80/8 mg/kg i.p.) to permit isolation of ventral tail arteries. Each artery was carefully sectioned into multiple 3 mm rings. In each experiment, half the rings from each vessel were pretreated for 10 minutes with 6-OHDA by a procedure known to remove sympathetic nerve endings without altering functional properties of the underlying smooth muscle [3]. Preliminary work confirmed the absence of nerve endings by failure of the rings to contract in response to tyramine, as expected [4]. Before experimentation, each ring was equilibrated at a passive tension of one gram in a bath containing 40 ml of
J.M. Lee, J.D. Peuler / Life Sciences 69 (2001) 1085–1092
1087
normal physiological buffer warmed to 378C and gassed to pH 7.4 with O2/CO2 (95/5%). Some of the rings scheduled to be contracted with exogenous NE before relaxation with metformin were first treated with iproniazid (to block monoamine oxidase) and/or desipramine (to block the neuronal NE carrier/reuptake pump). Some of the rings scheduled for contraction with phenylephrine (PE) before metformin were also first treated with desipramine. Some of the rings scheduled for contraction with 5-hydroxytryptamine (5HT) before metformin (leaving smooth muscle a-adrenergic receptors free to be stimulated by endogenous NE released from nerve endings) were first treated with phentolamine to block all a-adrenergic receptors. Each ring was then contracted with multiple concentrations of the scheduled contractile agonist to obtain its half-maximally effective concentration (EC50) for contracting that particular ring. After rinsing and re-equilibrating each ring in fresh buffer (free of the contractile agonist and any of the abovementioned experimental blockers) it was treated again with the same blocker (if scheduled) and contracted again with the same contractile agonist but only at its EC50 level. After the resultant half-maximal contraction stabilized, multiple levels of metformin were added cumulatively (0.12 to 22 mmol/L); 2-minutes per level. Acute relaxant responses to metformin were recorded at each level and its EC50 value for relaxation was computed (if possible). All such data were subjected to analysis-of-variance followed by multiple mean comparisons to detect statistically significant effects associated with the presence (or absence) of intact sympathetic nerve endings and the impact of any of the abovementioned experimental blocking agents. Results In SHR tail arterial rings lacking sympathetic nerve endings, metformin at all levels $ 0.12 mmol/L significantly relaxed the smooth muscle whether contracted half-maximally with exogenous NE or PE (Figures 1 and 2). The relaxation response to each level of metformin was rapid, reaching completion in 1–2 minutes. At the highest concentration of metformin (22 mmol/L), nearly all adrenergic contractions were abolished. Metformin also rapidly relaxed tail arterial smooth muscle precontracted half-maximally with 5HT (Figure 3) although beginning at a somewhat higher concentration than 0.12 mmol/L. Computation of the EC50 value for metformin’s relaxation of 5HT contraction was not always possible. The presence of intact sympathetic nerve endings significantly attenuated metformin’s ability to relax the underlying smooth muscle whether precontracted half-maximally with NE, PE or 5HT (Figures 1A, 2A and 3A). This attenuating effect was evident not only at several individual concentrations of metformin (beginning at $ 0.3 mmol/L, Figure 2A) but also in terms of a notable shift to the right in its EC50 values specifically for relaxation of adrenergic contractions (Figures 1A and 2A). Phentolamine abolished the attenuating effect of the nerve endings on metformin’s relaxation of smooth muscle precontracted with 5HT (Figure 3B). Desipramine abolished the attenuating effect of the nerve endings on metformin’s relaxation of underlying smooth muscle precontracted with either NE or PE (Figures 1B and 2B). Iproniazid notably enhanced the attenuating effect of the nerve endings on metformin’s relaxation of smooth muscle precontracted with exogenous NE (Figure 1C) and desipramine abolished this enhancement (Figure 1D).
1088
J.M. Lee, J.D. Peuler / Life Sciences 69 (2001) 1085–1092
Fig. 1. Relaxation by metformin of SHR tail arterial rings precontracted half-maximally with norepinephrine (NE). Enhancing effect of 231024 mol/L iproniazid (a monoamine oxidase inhibitor) and blocking effect of 1027 mol/L desipramine (a neuronal NE-carrier inhibitor) on the attenuating action of sympathetic nerve endings.
Discussion In the present study brief pretreatment of tail arterial tissue (freshly isolated from female SHR) with 6-OHDA, to remove sympathetic nerve endings stored with endogenous NE, subsequently enhanced the ability of metformin to rapidly relax smooth muscle in the same tissue precontracted with exogenous NE (Figure 1A). This enhancement is not likely due to lasting effects of the 6-OHDA on the smooth muscle’s responsiveness to vasorelaxant substances in general. In previous work with 6-OHDA we have not seen such enhancement when similar tissues were relaxed with either nifedipine or nitroprusside [5], two agents also known to rapidly relax smooth muscle but by distinctly different mechanisms and by acting only directly on smooth muscle and not indirectly through effects on sympathetic nerve endings [6,7,8]. Thus, in the case of metformin this enhancement is most likely due to removal of an action of the drug on the nerve endings themselves to either 1) stimulate release of en-
J.M. Lee, J.D. Peuler / Life Sciences 69 (2001) 1085–1092
1089
Fig. 2. Relaxation by metformin of SHR tail arterial rings precontracted half-maximally with phenylephrine (PE). Blocking effect of 1027 mol/L desipramine (a neuronal NE-carrier inhibitor) on the attenuating action of sympathetic nerve endings.
dogenous NE from them or 2) decrease re-uptake of the exogenous NE into them. However, removal of these nerve endings by 6-OHDA in the present study enhanced metformin’s relaxation of contractions produced not only by exogenous NE (Figure 1A) but also by phenylephrine (Figure 2A) and 5HT (Figure 3A), two agents which are not taken up as readily into sympathetic nerve endings as NE [9]. Thus, it is more likely that metformin at the levels administered in these experiments is rapidly stimulating release of endogenous NE from the sympathetic nerve endings, which would then explain why their presence attenuates the drug’s ability to relax the underlying smooth muscle (Figures 1A, 2A and 3A). This would
Fig. 3. Relaxation by metformin of SHR tail arterial rings precontracted half-maximally with 5-hydroxytryptamine (5HT). Blocking effect of 2.5x1027 mol/L phentolamine (an alpha-adrenergic receptor inhibitor) on the attenuating action of sympathetic nerve endings.
1090
J.M. Lee, J.D. Peuler / Life Sciences 69 (2001) 1085–1092
also explain why the attenuating effect of these nerve endings on metformin’s ability to relax smooth muscle precontracted specifically with 5HT was successfully abolished with phentolamine, a nonselective inhibitor of a-adrenergic receptors. It might appear from Figure 1A that metformin’s ability to release NE from sympathetic nerve endings is relatively minor compared to its overall ability to relax smooth muscle in tail arterial tissue from female SHR. However, in our previous in vivo work with this animal [2] total circulating endogenous NE was notably increased within one minute after bolus iv injection of metformin at 100 mg/kg (a dose likely to produce an immediate extracellular concentration of the drug near its EC50 values for tail arterial tissue as shown in Figure 1A). That systemic increase in circulating NE was observed with and without intact postganglionic sympathetic nerve action potentials (i.e. in the absence as well as in the presence of a ganglionic blocking agent) [2]. Thus, it was not reflexly mediated. It was also observed with and without the presence of intact adrenomedullary tissue [2]. Thus, it most likely originated from the vasculature at large and possibly the heart but certainly not just from the tail artery by itself. Thus, metformin’s NE-releasing action is likely a systemic event in female SHR and as such might have more than minor consequences. For example, in the same previous work there was evidence that this systemic release of NE participated in buffering metformin’s ability to decrease systemic arterial pressure [2]. Conceivably, it could also participate in buffering the drug’s systemic metabolic effects although that remains completely unexplored. Also, without more exploration we can only speculate on whether a systemic NE-releasing action occurs in males as well as females and in arterial tissue from normotensive as well as hypertensive animals. Some of our previous and current preliminary work (not shown here) would suggest that it might but such work is not yet complete. The mechanism responsible for the neural action of metformin as seen in the present study is not likely stimulation of exocytotic release of vesicularly-bound endogenous NE from the nerve ending directly into the synaptic cleft. That would require depolarization of the neuronal cell membrane. Others have demonstrated that metformin administered in vitro to the same arterial tissue at the same concentrations does not depolarize but rather repolarizes the smooth muscle cell membranes [10], an action which likely explains the drug’s direct relaxant effect. Thus, we chose to test the possibility that metformin may be acting like indirect sympathomimetic amines, agents that release NE from sympathetic nerve endings independent of exocytotic mechanisms [11–13]. Briefly, such agents release NE by first entering the nerve ending and interacting with storage vesicles, causing them to release significant amounts of their NE into the cytoplasm [11]. Then, as the concentration of free NE builds up in the cytoplasm the NE-carrier molecules in the neuronal cell membrane (that normally function as NE reuptake pumps) reverse their direction and transport the cytoplasmic NE out to the synaptic cleft [11,12]. While mechanisms of cell entry and vesicular interactions vary from one agent to another [11], they all share the latter step in common. Thus, other agents that can specifically inhibit all NE-carrier movement across cell membranes (in either direction), e.g. desipramine and other so-called NE reuptake inhibitors, are able to block the NEreleasing action of indirect sympathomimetic agents [11,12,13]. On the other hand, the drug iproniazid and other monoamine oxidase (MAO) inhibitors are able to enhance the action of indirect sympathomimetics because they block the metabolic inactivation of the free cytoplasmic NE by that enzyme, which permits greater buildup of that NE [11,12,13]. Con-
J.M. Lee, J.D. Peuler / Life Sciences 69 (2001) 1085–1092
1091
versely, neither the carrier (reuptake) inhibitors nor the MAO inhibitors have any meaningful effect on exocytotic release of vesicular NE [12,13]. Thus, since we found 1) that the abovementioned attenuating effect of sympathetic nerve endings on metformin’s relaxant action was considerably enhanced by iproniazid (Figure 1C) and 2) that this enhancing effect of MAO inhibition (plus the attenuating effect of the sympathetic nerve endings alone) was abolished by desipramine (Figure 1B and 1D), we conclude that the mechanism whereby metformin stimulates release of NE from sympathetic nerve endings is similar to that of indirect-acting sympathomimetic amines. Finally, the concentrations of metformin capable of exerting the acute effects seen in the present study are somewhat higher than those typically found in plasma (# 0.1 mmol/L; or # 100 micromolar) of either human patients or animal models following oral administration of therapeutically relevant doses [14,15]. However, it is also known that within a matter of hours after such standard oral dosing the drug can accumulate in various tissues up to concentrations several times higher than that present in plasma [14,15]. This could result in delayed actions of the drug which mimic actions that occur immediately upon exposure to higher levels. Thus, over long periods of time (typical of chronic oral dosing), we would expect to see evidence of some of the rapid direct actions observed in the present study. Such evidence now exists at least in terms of the effects of metformin on the arterial smooth muscle. In rats, long-term oral metformin has been shown to reduce intrinsic contractile reactivity of mesenteric arterial smooth muscle to NE ex vivo (i.e. even after such tissues are isolated from the animals) [16]. In humans, long-term oral metformin has been shown to reduce both systemic arterial pressures [reviewed in 17] and pressor responses to intravenously infused NE [18]. Thus, the acute in vitro relaxant effect of the drug in the present study on arterial smooth muscle may well reflect a mechanism underlying its long-term antihypertensive action after chronic oral administration of clinically-relevant doses to diabetic patients [17]. However, as yet there have been no efforts to determine whether the potential indirect sympathomimetic action of metformin (described herein) also occurs in diabetic patients, possibly buffering the drug’s antihypertensive effectiveness following long-term oral administration. More importantly, there have been no efforts to look for evidence that antidepressant drugs with properties similar to iproniazid and desipramine might interact with metformin to alter its antihypertensive effectiveness in such patients. Such a goal is now clearly worthy of careful investigation in light of recent increasing awareness that depression frequently co-exists with type 2 diabetes in the same patients due to common underlying genetic and environmental causes [reviewed in 19 and 20]. Acknowledgments The authors wish to acknowledge the excellent secretarial assistance of Victoria L. Sears of Midwestern University. This study was funded by a grant from the NIH (#1-R15-HL/62238). References 1. Peuler JD. Adrenergic depressor and pressor actions of metformin in spontaneously hypertensive rats [abstract]. Am J Hypertens 1997;10:107A.
1092
J.M. Lee, J.D. Peuler / Life Sciences 69 (2001) 1085–1092
2. Peuler, JD. Opposing adrenergic actions of intravenous metformin on arterial pressure in female spontaneously hypertensive rats. Cardiovascular Research 1999;43:237–247. 3. Aprigliano O, Hermsmeyer K. In vitro denervation of the portal vein and caudal artery of the rat. J Pharmacol Exp Ther 1976;198:568–577. 4. Webb RC, Vanhoutte PM, Bohr DF. Adrenergic neurotransmission in vascular smooth muscle from spontaneously hypertensive rats. Hypertension 1981;3:93–103. 5. Lee JM, Peuler JD. Acute vasorelaxant effects of metformin and attenuation by stimulation of sympathetic agonist release. Life Sciences 1998;64(4):PL57–PL63. 6. Friedman DJ, Duckles SP. Effect of calcium channel blockers on norepinephrine release and modulation by prejunctional D2 dopamine receptors. Life Sciences 1994;54:1545–1557. 7. Göthert M, Molderings GJ. Mibefradil- and omega-conotoxin GVIA-induced inhibition of noradrenaline release from the sympathetic nerves of the human heart. Naunyn-Schmiedeberg’s Arch Pharmacol 1997;356: 860–863. 8. Hunter LW, Tyce GM, Rorie DK: Norepinephrine release during vasoconstriction induced by cross-linked hemoglobin. Life Sciences 1996;59:131–140. 9. Iverson, LL. The Uptake and Storage of Noradrenaline in Sympathetic Nerves. Cambridge: The University Press, 1967. pp. 147–198. 10. Chen X-L, Panek K, Rembold CM. Metformin relaxes rat tail artery by repolarization and resultant decreases in Ca21 influx and intracellular [Ca21]. J Hypertens 1997;15:269–274. 11. Raiteri M, Levi G. Reviews of Neuroscience, Volume 3. New York: Raven Press, 1978. pp. 101–111. 12. Vanhoutte PM, Verbeuren TJ, Webb RC. Local modulation of adrenergic neuroeffector interaction in the blood vessel wall. Physiological Reviews 1981;61:151–247. 13. Hayashi S, Park MK. Effects of nialamide on responses of dog isolated arteries to tyramine and transmural electrical stimulation. Gen Pharmac 1984;15:19–24. 14. Sirtori CR, Franceshini G, Galli-Kienle M, Cighetti G, Galli G, Bondioli A, Conti F. Disposition of metformin (N,N-dimethylbiguanide) in man. Clin Pharmacol Ther 1978;24:683–693. 15. Wilcock C, Bailey CJ. Accumulation of metformin by tissues of the normal and diabetic mouse. Xenobiotica 1994;24:49–57. 16. Verma S, Bhanot S, McNeill JH. Decreased vascular reactivity in metformin-treated fructose-hypertensive rats. Metabolism 1996;45:1053–1055. 17. Howes LG, Sundaresan P, Lykos D. Cardiovascular effects of oral hypoglycemic drugs. Clin Exp Pharmacol Physiol 1996;23:201–206. 18. Sundaresan P, Lykos D, Daher A, Diamond T, Morris R, Howes LG. Comparative effects of glibenclamide and metformin on ambulatory blood pressure and cardiovascular reactivity in NIDDM. Diabetes Care 1997;20:692–697. 19. Okamura F, Tashiro A, Utsumi A, Imai T, Suchi T, Hongo M. Insulin resistance in patients with depression and its changes in the clinical course of depression: a report on three cases using the minimal model analysis. Intern Med 1999; 38:357–360. 20. Björntorp P, Holm G, Rosmond R. Hypothalamic arousal, insulin resistance and type 2 diabetes mellitus. Diabet Med 1999;16: 373–383.
A possible indirect sympathomimetic action of metformin in the arterial vessel wall of spontanously hypertensive rats JiHun M. Lee, Jacob D. Peuler* Department of Pharmacology, Midwestern University, 555 31st Street, Downers Grove, IL 60515, USA Received 7 August 2000; accepted 30 January 2001
Abstract The antidiabetic drug metformin (MF) typically achieves only micromolar levels in plasma with normal therapeutic use. However, it is also known to accumulate in various tissues up to several times higher after standard oral dosing and we now have evidence from both in vivo and in vitro experiments with spontaneously hypertensive rats (SHR) that millimolar levels stimulate release of norepinephrine (NE) from vascular sympathetic nerve endings (SNEs). As shown in the present work with SHR tail arterial tissue (rich in SNEs), the known vasodilator effect of millimolar levels of MF on the smooth muscle (even if contracted with a nonadrenergic agonist), is attenuated by the presence of the SNEs unless phentolamine (an alpha receptor blocker) is present. We reasoned that the mechanism for this apparent NE-releasing action of MF is not exocytotic release as that would require depolarization of the neuronal cell membranes in SNEs, and MF at millimolar levels is known to repolarize (not depolarize) membranes of other cells. Thus, we tested the possibility that MF releases NE by an indirect sympathomimetic-like action. Such an action should be amplified by monoamine oxidase inhibitors (e.g. iproniazid) and blocked by NE-carrier inhibitors (e.g. desipramine). Accordingly, we found that the abovementioned attenuating effect of intact SNEs on MF’s relaxation of SHR tail arterial tissue (compared to tissues in which SNEs were experimentally removed with 6-hydroxydopamine) was amplified nearly 3-fold by iproniazid (p,0.05) and blocked by desipramine (p,0.05). These results support an indirect sympathomimetic action of MF and raise the question whether commonly used antidepressants with properties similar to iproniazid and desipramine might alter MF’s beneficial vasodilatory (and thus antihypertensive) effectiveness in diabetic patients with hypertension. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Norepinephrine; Adrenergic nerve endings; Vasorelaxation; Diabetes; Antidepressants
* Corresponding author. Tel.: (630) 515-6068; fax: (630) 971-6414. E-mail address: [email protected] (J.D. Peuler) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 2 0 2 -4
1086
J.M. Lee, J.D. Peuler / Life Sciences 69 (2001) 1085–1092
Introduction We have found that the antidiabetic drug metformin at concentrations above 0.1 mmol/L rapidly relaxes adrenergic contractions in vitro in intact vascular tissue rings prepared from the ventral tail artery of the female spontaneously hypertensive rat (SHR) [1]. This acute relaxant effect is not likely limited to just the tail artery as we have also found that bolus intravenous (iv) injection of metformin (at doses likely to produce such concentrations in plasma) rapidly decreases not only resting arterial pressures in such SHR but also pressures supported specifically by iv infusion of exogenous a-adrenergic agonists [2]. However, these acute depressor responses to iv metformin in these same SHR were accompanied by immediate increases in arterial plasma norepinephrine (NE) levels; increases not dependent on the presence of the adrenomedulla and seen in ganglionically-blocked as well as autonomically-intact animals [2]. Thus, we know that they are neither adrenomedullary in origin nor reflexly-mediated. Rather, we concluded from that in vivo work that metformin can directly stimulate release of NE from postganglionic sympathetic nerve endings independent of axonal action potentials [2]. However, we were not able to determine in an in vivo study of that type whether such a neural action of metformin was at all functionally important in the arterial vessel wall. For example, does it compromise the direct relaxant action of the drug on the underlying smooth muscle? The ventral tail artery of the rat is rich in sympathetic nerve endings as well as smooth muscle cells [3,4]. Thus we decided to re-examine the abovementioned acute relaxant effect of metformin on adrenergic contractions in vitro in tail arterial tissue isolated from female SHR but this time after its pretreatment with 6-hydroxydopamine (6-OHDA) which specifically removes sympathetic nerve endings without altering the ability of the underlying smooth muscle to either contract or relax [3,5]. We hypothesized that this pretreatment would notably enhance metformin’s relaxant effect or, conversely, that the presence of intact sympathetic nerve endings would notably attenuate its relaxant effect. Indeed, such results were observed and thus we conducted additional experiments in an attempt to ascertain the mechanism responsible for this effect. We focused on the possibility that metformin may be releasing NE from nerve endings by way of an indirect sympathomimetic-like action. Thus, as expected with such an action, we hypothesized that a monoamine oxidase (MAO) inhibitor would enhance it (i.e. enhance the attenuating effect of the nerve endings on metformin’s relaxation) and a NE-carrier inhibitor would abolish it. Methods As in the previous work that led up to this study [2], we used adult female SHR in the present experiments. They were housed as described previously under conditions known to desynchronize estrous cycles [2]. Then they were selected at random (one rat per day) and anesthetized with ketamine/xylazine (80/8 mg/kg i.p.) to permit isolation of ventral tail arteries. Each artery was carefully sectioned into multiple 3 mm rings. In each experiment, half the rings from each vessel were pretreated for 10 minutes with 6-OHDA by a procedure known to remove sympathetic nerve endings without altering functional properties of the underlying smooth muscle [3]. Preliminary work confirmed the absence of nerve endings by failure of the rings to contract in response to tyramine, as expected [4]. Before experimentation, each ring was equilibrated at a passive tension of one gram in a bath containing 40 ml of
J.M. Lee, J.D. Peuler / Life Sciences 69 (2001) 1085–1092
1087
normal physiological buffer warmed to 378C and gassed to pH 7.4 with O2/CO2 (95/5%). Some of the rings scheduled to be contracted with exogenous NE before relaxation with metformin were first treated with iproniazid (to block monoamine oxidase) and/or desipramine (to block the neuronal NE carrier/reuptake pump). Some of the rings scheduled for contraction with phenylephrine (PE) before metformin were also first treated with desipramine. Some of the rings scheduled for contraction with 5-hydroxytryptamine (5HT) before metformin (leaving smooth muscle a-adrenergic receptors free to be stimulated by endogenous NE released from nerve endings) were first treated with phentolamine to block all a-adrenergic receptors. Each ring was then contracted with multiple concentrations of the scheduled contractile agonist to obtain its half-maximally effective concentration (EC50) for contracting that particular ring. After rinsing and re-equilibrating each ring in fresh buffer (free of the contractile agonist and any of the abovementioned experimental blockers) it was treated again with the same blocker (if scheduled) and contracted again with the same contractile agonist but only at its EC50 level. After the resultant half-maximal contraction stabilized, multiple levels of metformin were added cumulatively (0.12 to 22 mmol/L); 2-minutes per level. Acute relaxant responses to metformin were recorded at each level and its EC50 value for relaxation was computed (if possible). All such data were subjected to analysis-of-variance followed by multiple mean comparisons to detect statistically significant effects associated with the presence (or absence) of intact sympathetic nerve endings and the impact of any of the abovementioned experimental blocking agents. Results In SHR tail arterial rings lacking sympathetic nerve endings, metformin at all levels $ 0.12 mmol/L significantly relaxed the smooth muscle whether contracted half-maximally with exogenous NE or PE (Figures 1 and 2). The relaxation response to each level of metformin was rapid, reaching completion in 1–2 minutes. At the highest concentration of metformin (22 mmol/L), nearly all adrenergic contractions were abolished. Metformin also rapidly relaxed tail arterial smooth muscle precontracted half-maximally with 5HT (Figure 3) although beginning at a somewhat higher concentration than 0.12 mmol/L. Computation of the EC50 value for metformin’s relaxation of 5HT contraction was not always possible. The presence of intact sympathetic nerve endings significantly attenuated metformin’s ability to relax the underlying smooth muscle whether precontracted half-maximally with NE, PE or 5HT (Figures 1A, 2A and 3A). This attenuating effect was evident not only at several individual concentrations of metformin (beginning at $ 0.3 mmol/L, Figure 2A) but also in terms of a notable shift to the right in its EC50 values specifically for relaxation of adrenergic contractions (Figures 1A and 2A). Phentolamine abolished the attenuating effect of the nerve endings on metformin’s relaxation of smooth muscle precontracted with 5HT (Figure 3B). Desipramine abolished the attenuating effect of the nerve endings on metformin’s relaxation of underlying smooth muscle precontracted with either NE or PE (Figures 1B and 2B). Iproniazid notably enhanced the attenuating effect of the nerve endings on metformin’s relaxation of smooth muscle precontracted with exogenous NE (Figure 1C) and desipramine abolished this enhancement (Figure 1D).
1088
J.M. Lee, J.D. Peuler / Life Sciences 69 (2001) 1085–1092
Fig. 1. Relaxation by metformin of SHR tail arterial rings precontracted half-maximally with norepinephrine (NE). Enhancing effect of 231024 mol/L iproniazid (a monoamine oxidase inhibitor) and blocking effect of 1027 mol/L desipramine (a neuronal NE-carrier inhibitor) on the attenuating action of sympathetic nerve endings.
Discussion In the present study brief pretreatment of tail arterial tissue (freshly isolated from female SHR) with 6-OHDA, to remove sympathetic nerve endings stored with endogenous NE, subsequently enhanced the ability of metformin to rapidly relax smooth muscle in the same tissue precontracted with exogenous NE (Figure 1A). This enhancement is not likely due to lasting effects of the 6-OHDA on the smooth muscle’s responsiveness to vasorelaxant substances in general. In previous work with 6-OHDA we have not seen such enhancement when similar tissues were relaxed with either nifedipine or nitroprusside [5], two agents also known to rapidly relax smooth muscle but by distinctly different mechanisms and by acting only directly on smooth muscle and not indirectly through effects on sympathetic nerve endings [6,7,8]. Thus, in the case of metformin this enhancement is most likely due to removal of an action of the drug on the nerve endings themselves to either 1) stimulate release of en-
J.M. Lee, J.D. Peuler / Life Sciences 69 (2001) 1085–1092
1089
Fig. 2. Relaxation by metformin of SHR tail arterial rings precontracted half-maximally with phenylephrine (PE). Blocking effect of 1027 mol/L desipramine (a neuronal NE-carrier inhibitor) on the attenuating action of sympathetic nerve endings.
dogenous NE from them or 2) decrease re-uptake of the exogenous NE into them. However, removal of these nerve endings by 6-OHDA in the present study enhanced metformin’s relaxation of contractions produced not only by exogenous NE (Figure 1A) but also by phenylephrine (Figure 2A) and 5HT (Figure 3A), two agents which are not taken up as readily into sympathetic nerve endings as NE [9]. Thus, it is more likely that metformin at the levels administered in these experiments is rapidly stimulating release of endogenous NE from the sympathetic nerve endings, which would then explain why their presence attenuates the drug’s ability to relax the underlying smooth muscle (Figures 1A, 2A and 3A). This would
Fig. 3. Relaxation by metformin of SHR tail arterial rings precontracted half-maximally with 5-hydroxytryptamine (5HT). Blocking effect of 2.5x1027 mol/L phentolamine (an alpha-adrenergic receptor inhibitor) on the attenuating action of sympathetic nerve endings.
1090
J.M. Lee, J.D. Peuler / Life Sciences 69 (2001) 1085–1092
also explain why the attenuating effect of these nerve endings on metformin’s ability to relax smooth muscle precontracted specifically with 5HT was successfully abolished with phentolamine, a nonselective inhibitor of a-adrenergic receptors. It might appear from Figure 1A that metformin’s ability to release NE from sympathetic nerve endings is relatively minor compared to its overall ability to relax smooth muscle in tail arterial tissue from female SHR. However, in our previous in vivo work with this animal [2] total circulating endogenous NE was notably increased within one minute after bolus iv injection of metformin at 100 mg/kg (a dose likely to produce an immediate extracellular concentration of the drug near its EC50 values for tail arterial tissue as shown in Figure 1A). That systemic increase in circulating NE was observed with and without intact postganglionic sympathetic nerve action potentials (i.e. in the absence as well as in the presence of a ganglionic blocking agent) [2]. Thus, it was not reflexly mediated. It was also observed with and without the presence of intact adrenomedullary tissue [2]. Thus, it most likely originated from the vasculature at large and possibly the heart but certainly not just from the tail artery by itself. Thus, metformin’s NE-releasing action is likely a systemic event in female SHR and as such might have more than minor consequences. For example, in the same previous work there was evidence that this systemic release of NE participated in buffering metformin’s ability to decrease systemic arterial pressure [2]. Conceivably, it could also participate in buffering the drug’s systemic metabolic effects although that remains completely unexplored. Also, without more exploration we can only speculate on whether a systemic NE-releasing action occurs in males as well as females and in arterial tissue from normotensive as well as hypertensive animals. Some of our previous and current preliminary work (not shown here) would suggest that it might but such work is not yet complete. The mechanism responsible for the neural action of metformin as seen in the present study is not likely stimulation of exocytotic release of vesicularly-bound endogenous NE from the nerve ending directly into the synaptic cleft. That would require depolarization of the neuronal cell membrane. Others have demonstrated that metformin administered in vitro to the same arterial tissue at the same concentrations does not depolarize but rather repolarizes the smooth muscle cell membranes [10], an action which likely explains the drug’s direct relaxant effect. Thus, we chose to test the possibility that metformin may be acting like indirect sympathomimetic amines, agents that release NE from sympathetic nerve endings independent of exocytotic mechanisms [11–13]. Briefly, such agents release NE by first entering the nerve ending and interacting with storage vesicles, causing them to release significant amounts of their NE into the cytoplasm [11]. Then, as the concentration of free NE builds up in the cytoplasm the NE-carrier molecules in the neuronal cell membrane (that normally function as NE reuptake pumps) reverse their direction and transport the cytoplasmic NE out to the synaptic cleft [11,12]. While mechanisms of cell entry and vesicular interactions vary from one agent to another [11], they all share the latter step in common. Thus, other agents that can specifically inhibit all NE-carrier movement across cell membranes (in either direction), e.g. desipramine and other so-called NE reuptake inhibitors, are able to block the NEreleasing action of indirect sympathomimetic agents [11,12,13]. On the other hand, the drug iproniazid and other monoamine oxidase (MAO) inhibitors are able to enhance the action of indirect sympathomimetics because they block the metabolic inactivation of the free cytoplasmic NE by that enzyme, which permits greater buildup of that NE [11,12,13]. Con-
J.M. Lee, J.D. Peuler / Life Sciences 69 (2001) 1085–1092
1091
versely, neither the carrier (reuptake) inhibitors nor the MAO inhibitors have any meaningful effect on exocytotic release of vesicular NE [12,13]. Thus, since we found 1) that the abovementioned attenuating effect of sympathetic nerve endings on metformin’s relaxant action was considerably enhanced by iproniazid (Figure 1C) and 2) that this enhancing effect of MAO inhibition (plus the attenuating effect of the sympathetic nerve endings alone) was abolished by desipramine (Figure 1B and 1D), we conclude that the mechanism whereby metformin stimulates release of NE from sympathetic nerve endings is similar to that of indirect-acting sympathomimetic amines. Finally, the concentrations of metformin capable of exerting the acute effects seen in the present study are somewhat higher than those typically found in plasma (# 0.1 mmol/L; or # 100 micromolar) of either human patients or animal models following oral administration of therapeutically relevant doses [14,15]. However, it is also known that within a matter of hours after such standard oral dosing the drug can accumulate in various tissues up to concentrations several times higher than that present in plasma [14,15]. This could result in delayed actions of the drug which mimic actions that occur immediately upon exposure to higher levels. Thus, over long periods of time (typical of chronic oral dosing), we would expect to see evidence of some of the rapid direct actions observed in the present study. Such evidence now exists at least in terms of the effects of metformin on the arterial smooth muscle. In rats, long-term oral metformin has been shown to reduce intrinsic contractile reactivity of mesenteric arterial smooth muscle to NE ex vivo (i.e. even after such tissues are isolated from the animals) [16]. In humans, long-term oral metformin has been shown to reduce both systemic arterial pressures [reviewed in 17] and pressor responses to intravenously infused NE [18]. Thus, the acute in vitro relaxant effect of the drug in the present study on arterial smooth muscle may well reflect a mechanism underlying its long-term antihypertensive action after chronic oral administration of clinically-relevant doses to diabetic patients [17]. However, as yet there have been no efforts to determine whether the potential indirect sympathomimetic action of metformin (described herein) also occurs in diabetic patients, possibly buffering the drug’s antihypertensive effectiveness following long-term oral administration. More importantly, there have been no efforts to look for evidence that antidepressant drugs with properties similar to iproniazid and desipramine might interact with metformin to alter its antihypertensive effectiveness in such patients. Such a goal is now clearly worthy of careful investigation in light of recent increasing awareness that depression frequently co-exists with type 2 diabetes in the same patients due to common underlying genetic and environmental causes [reviewed in 19 and 20]. Acknowledgments The authors wish to acknowledge the excellent secretarial assistance of Victoria L. Sears of Midwestern University. This study was funded by a grant from the NIH (#1-R15-HL/62238). References 1. Peuler JD. Adrenergic depressor and pressor actions of metformin in spontaneously hypertensive rats [abstract]. Am J Hypertens 1997;10:107A.
1092
J.M. Lee, J.D. Peuler / Life Sciences 69 (2001) 1085–1092
2. Peuler, JD. Opposing adrenergic actions of intravenous metformin on arterial pressure in female spontaneously hypertensive rats. Cardiovascular Research 1999;43:237–247. 3. Aprigliano O, Hermsmeyer K. In vitro denervation of the portal vein and caudal artery of the rat. J Pharmacol Exp Ther 1976;198:568–577. 4. Webb RC, Vanhoutte PM, Bohr DF. Adrenergic neurotransmission in vascular smooth muscle from spontaneously hypertensive rats. Hypertension 1981;3:93–103. 5. Lee JM, Peuler JD. Acute vasorelaxant effects of metformin and attenuation by stimulation of sympathetic agonist release. Life Sciences 1998;64(4):PL57–PL63. 6. Friedman DJ, Duckles SP. Effect of calcium channel blockers on norepinephrine release and modulation by prejunctional D2 dopamine receptors. Life Sciences 1994;54:1545–1557. 7. Göthert M, Molderings GJ. Mibefradil- and omega-conotoxin GVIA-induced inhibition of noradrenaline release from the sympathetic nerves of the human heart. Naunyn-Schmiedeberg’s Arch Pharmacol 1997;356: 860–863. 8. Hunter LW, Tyce GM, Rorie DK: Norepinephrine release during vasoconstriction induced by cross-linked hemoglobin. Life Sciences 1996;59:131–140. 9. Iverson, LL. The Uptake and Storage of Noradrenaline in Sympathetic Nerves. Cambridge: The University Press, 1967. pp. 147–198. 10. Chen X-L, Panek K, Rembold CM. Metformin relaxes rat tail artery by repolarization and resultant decreases in Ca21 influx and intracellular [Ca21]. J Hypertens 1997;15:269–274. 11. Raiteri M, Levi G. Reviews of Neuroscience, Volume 3. New York: Raven Press, 1978. pp. 101–111. 12. Vanhoutte PM, Verbeuren TJ, Webb RC. Local modulation of adrenergic neuroeffector interaction in the blood vessel wall. Physiological Reviews 1981;61:151–247. 13. Hayashi S, Park MK. Effects of nialamide on responses of dog isolated arteries to tyramine and transmural electrical stimulation. Gen Pharmac 1984;15:19–24. 14. Sirtori CR, Franceshini G, Galli-Kienle M, Cighetti G, Galli G, Bondioli A, Conti F. Disposition of metformin (N,N-dimethylbiguanide) in man. Clin Pharmacol Ther 1978;24:683–693. 15. Wilcock C, Bailey CJ. Accumulation of metformin by tissues of the normal and diabetic mouse. Xenobiotica 1994;24:49–57. 16. Verma S, Bhanot S, McNeill JH. Decreased vascular reactivity in metformin-treated fructose-hypertensive rats. Metabolism 1996;45:1053–1055. 17. Howes LG, Sundaresan P, Lykos D. Cardiovascular effects of oral hypoglycemic drugs. Clin Exp Pharmacol Physiol 1996;23:201–206. 18. Sundaresan P, Lykos D, Daher A, Diamond T, Morris R, Howes LG. Comparative effects of glibenclamide and metformin on ambulatory blood pressure and cardiovascular reactivity in NIDDM. Diabetes Care 1997;20:692–697. 19. Okamura F, Tashiro A, Utsumi A, Imai T, Suchi T, Hongo M. Insulin resistance in patients with depression and its changes in the clinical course of depression: a report on three cases using the minimal model analysis. Intern Med 1999; 38:357–360. 20. Björntorp P, Holm G, Rosmond R. Hypothalamic arousal, insulin resistance and type 2 diabetes mellitus. Diabet Med 1999;16: 373–383.