The potential antidiabetic activity of some alpha-2 adrenoceptor antagonists

Pharmacological Research, Vol. 44, No. 5, 2001 doi:10.1006/phrs.2001.0870, available online at http://www.idealibrary.com on

THE POTENTIAL ANTIDIABETIC ACTIVITY OF SOME ALPHA-2 ADRENOCEPTOR ANTAGONISTS AHMED O. ABDEL-ZAHER∗ , IHAB T. AHMED and ALAA EL-DIN A. EL-KOUSSI Department of Pharmacology, Faculty of Medicine, Assiut University, Assiut, Egypt Accepted 31 July 2001

The effect of alpha-2 adrenoceptor antagonists, yohimbine and efaroxan, on the plasma glucose and insulin levels was studied in non-diabetic control, type-I (insulin-dependent) and type-II (non-insulin-dependent) diabetic rats. Pretreatment with either yohimbine or efaroxan potentiated glucose-induced insulin release in non-diabetic control rats and produced an improvement of the oral glucose tolerance and potentiated glucose-induced insulin release in type-II but not in type-I diabetic rats. Treatment with either yohimbine or efaroxan reduced the plasma glucose level and increased the plasma insulin level of non-diabetic control and type-II diabetic rats but not of type-I diabetic rats. Effects of efaroxan were more marked. Pretreatment of non-diabetic control and typeII diabetic rats with either yohimbine or efaroxan inhibited clonidine-induced hyperglycaemia and suppressed or reversed clonidine-induced hypoinsulinaemia. Also, pretreatment of these animals with either yohimbine or efaroxan enhanced the hypoglycaemic and insulinotropic effects of glibenclamide. The combination of glibenclamide and efaroxan led to a synergistic increase in insulin secretion, while that of glibenclamide and yohimbine led to an additive increase. The hyperglycaemic effect of diazoxide in non-diabetic control and type-II diabetic rats was inhibited by pretreatment with either yohimbine or efaroxan. The hypoinsulinaemic effect of diazoxide in these animals was antagonized and reversed by pretreatment with yohimbine and efaroxan, respectively. In type-I diabetic rats, there was no change in the plasma glucose and insulin levels induced by the treatment of animals with each of clonidine or diazoxide alone or in combination with either yohimbine or efaroxan. Glibenclamide produced a slight decrease in the plasma glucose level of type-I diabetic rats, at the end of the 120 min period of investigation but there was no change in the plasma insulin level. Pretreatment of these animals with either yohimbine or efaroxan produced no change in glibenclamide effects. Additionally, bath application of efaroxan or glibenclamide inhibited the relaxant effects of different concentrations of diazoxide on the isolated norepinephrinecontracted aortic strips, while the application of yohimbine produced insignificant changes. The combination of glibenclamide and efaroxan led to complete inhibition of the relaxant effects of different concentrations of diazoxide, while that of glibenclamide and yohimbine did not produce such an effect. It is concluded that yohimbine, via blockade of postsynaptic alpha-2 adrenoceptors, and efaroxan, via blockade of postsynaptic alpha-2 adrenoceptors and adenosine triphosphatesensitive potassium channels in the pancreatic beta-cell membrane, produce insulinotropic and c 2001 Academic Press subsequent hypoglycaemic effects.

K EY WORDS : yohimbine, efaroxan, type-I diabetic rats, type-II diabetic rats, glibenclamide.

INTRODUCTION The control of insulin secretion by the sympathetic nervous system and circulating catecholamines is dependent on a functional balance between alpha-2 and beta-2 adrenoceptor activities. Stimulation of alpha-2 adrenoceptors on pancreatic beta-cells was ∗ Corresponding author.

1043–6618/01/110397–13/$35.00/0

found to inhibit insulin secretion, whereas stimulation of beta-2 adrenoceptors was found to enhance insulin secretion [1–3]. Some studies have shown that certain alpha-2 adrenoceptor antagonists are able to enhance the rate of insulin secretion when administered to experimental animals or human volunteers in vivo or when added to isolated islets of Langerhans incubated in vitro [4, 5]. These effects have been attributed to blockade of islet alpha-2 adrenocepc 2001 Academic Press

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tors [6, 7] leading to an increase in insulin secretion by virtue of the relief of the inhibitory tone mediated by endogenous catecholamines [8]. Based on these findings, it has been postulated that alpha-2 adrenoceptor antagonists could have potential in the treatment of type-II diabetes mellitus [6]. This concept has led to testing alpha-2 adrenoceptor antagonists as putative antidiabetic agents [6, 9]. However, the capacity of the alpha-2 adrenoceptor antagonists to enhance the rate of insulin secretion, cannot solely be attributed to blockade of islet alpha-2 adrenoceptors. Several lines of evidence indicate that alpha-2 adrenoceptor antagonists stimulate insulin secretion independently of the alpha-2 adrenoceptors blockade [2]. Also, in many cases the alpha-2 adrenoceptors blockade may not lead to the stimulation of insulin release [10, 11]. Stimulation of insulin secretion from pancreatic betacells is evoked physiologically by an elevation of the plasma glucose level, via metabolic production of adenosine triphosphate (ATP) which then inhibits the ATPsensitive potassium (KATP ) channels [12]. It has been established that the activity of the KATP channels is a major determinant of the resting potential of beta-cells. Inhibition of the channels results in membrane depolarization followed by an increase in the cytosolic Ca2+ concentration through Ca2+ entry via voltage-dependent Ca2+ channels and eventual exocytosis of insulin [12, 13]. It has been reported that the alpha-2 adrenoceptor antagonists that are imidazoline compounds stimulate insulin release independently of the alpha-2 adrenoceptors’ blocking property [14]. Rather, it is suggested that these drugs (e.g. efaroxan) stimulate insulin secretion by blocking KATP channels in pancreatic betacells [5, 8, 15, 16]. However, there is some controversy about which of these two possible mechanisms accounts for the ability of imidazoline compounds to enhance the insulin secretory effects [7, 8, 17]. In light of these observations, the objective of this work is to investigate the potential antidiabetic activity of some alpha-2 adrenoceptor antagonists. In addition, an attempt was undertaken to elucidate the possible mechanisms(s) of this antidiabetic activity of these drugs.

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single i.p. injection of 95 mg kg−1 streptozotocin. After weaning, rats were individually housed with water and food available ad libitum. When rats were 9 weeks old they were used to evaluate the effect of drugs under investigation. Control animals received only the vehicle.

Oral glucose tolerance test Groups, each of six animals, of non-diabetic control, type-I and type-II diabetic rats were fasted overnight before the day of experiments with free access to water. These animals were treated with an oral D-glucose load of 2 g kg−1 by means of a stomach tube. Also, in this set of experiments, similar groups of non-diabetic control, type-1 and type-II diabetic rats were treated with alpha-2 antagonists (20 mg kg−1 yohimbine i.p. of 5 mg kg−1 efaroxan orally by means of a stomach tube) 30 min before the oral administration of the D-glucose load. Control animals were treated likewise with the vehicle (isotonic saline). Blood samples were collected for glucose and insulin level estimations from the orbital sinus of rats before and 15, 30, 60 and 120 min after the administration of the oral D-glucose load. In this study, pentobarbital sodium was used to minimize the effect of the stress resulting from handling, injection and blood sampling on animals [20]. The i.p. injection of 25 mg kg−1 pentobarbital sodium into nondiabetic control type-I and type-II diabetic rats did not produce any significant changes in blood glucose and insulin levels of these animals.

Determination of the effect of alpha-2 adrenoceptor antagonists, yohimbine and efaroxan, on plasma glucose and insulin levels of non-diabetic control, type-I and type-II diabetic rats In these experiments, groups, each of six animals, of non-diabetic control, type-I and type-II diabetic rats were used. Animals were fasted overnight before the day of the experiment with free access to water. These animals were treated with either 20 mg kg−1 yohimbine i.p. or 5 mg kg−1 efaroxan orally. Control animals received an equal volume of the vehicle (isotonic saline). Blood samples were collected for glucose and insulin level estimations before and 15, 30, 60 and 120 min after each treatment.

MATERIALS AND METHODS

Induction of diabetes mellitus in rats Type-I diabetes (insulin-dependent diabetes mellitus) was induced by an intraperitoneal (i.p.) injection of 65 mg kg−1 streptozotocin in 9-week-old male Wistar rats [18]. Streptozotocin was dissolved in isotonic saline immediately before use. Animals were used to study the effect of drugs under investigation 2 days after the injection of streptozotocin. Control animals received only the vehicle. As described by Angel et al. [19], type-II diabetes (non-insulin-dependent diabetes mellitus) was induced in 1-day-old male Wistar rats by treating them with a

Determination of the effect of alpha-2 adrenoceptor antagonists, yohimbine and efaroxan, on clonidine, glibenclamide and diazoxide-induced alterations in plasma glucose and insulin levels of non-diabetic control, type-I and type-II diabetic rats The overnight fasted non-diabetic control, type-I and type-II diabetic rats were treated in these experiments with 300 µg kg−1 clonidine (in saline) subcutaneously, 5 mg kg−1 glibenclamide (in saline/NaOH/ethanol) orally or with 30 mg kg−1 diazoxide (in saline/NaOH/ ethanol) i.p. Control animals were treated likewise with the vehicle. Other groups of animals were treated with either 20 mg kg−1 yohimbine i.p., or 5 mg kg−1 efaroxan

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orally for 30 min before the subcutaneous injection of clonidine, oral administration of glibenclamide or i.p. injection of diazoxide. Blood samples were collected for glucose and insulin level estimations before and 15, 30, 60 and 120 min after treatment of rats with clonidine, glibenclamide or diazoxide.

contracted with norepinephrine were determined. One tissue always served as a control. The final concentration of ethanol in the bathing fluid was always less than 0.2% which had no effect on tissue responses.

In vitro studies

The plasma glucose level was assayed by an enzymatic colorimetric method using a commercially available Glucose Enzymatique PAP kit (Bio Merieux, France). The plasma insulin concentration was measured by the radioimmunoassay method using a commercially available Coat-A-Count Insulin kit (Diagnostic Products Corporation, USA)

Adult male rabbits weighing 1.5–2.5 kg were used in these experiments. Animals were killed by decapitation. In each rabbit, the thorax was opened and the descending aorta was removed. The aorta was immediately placed in ice-cold oxygenated Kerb’s-Henseleit solution. The composition of Kerb’s-Henseleit solution was as follows (mmol l−1 ): NaCl 118; KCl 4.69; CaCl2 2.52; NaHCO3 25; MgSO4 .7H2 O 1.18; KH2 PO4 1.17; and D -glucose 11.10. The vessel was cleaned of fat and loose connective tissue and cut into long continuous spiral strips (5 mm wide and 3–4 cm long, approximately) by threading a glass rod through the lumen of the aorta and with a pair of fine scissors. The aortic strips were suspended in an organ bath containing 20 ml of Kerb’sHenseleit solution maintained at 37 ◦ C and bubbled with carbogen mixture (5% CO2 in O2 ). The strips were connected to a T2 isotonic transducer and an amplifier of a two-channel Oscillograph MD2 (BioScience, Kent, UK). Each preparation was allowed to equilibrate for 30 min before the experiment was commenced, during which time the physiological salt solution was renewed every 15 min. In these experiments, the aortic strips were first contracted with 5 µmol l−1 norepinephrine (from a stock solution of 1 mmol l−1 in saline). Tissues were then washed several times and allowed to relax to base line levels, before the same concentration of norepinephrine was reapplied. At the plateau of the norepinephrine contraction, tissues were exposed to graded concentrations (0.01–1 mmol l−1 ) of diazoxide (from a stock solution of 5 mmol l−1 in saline/ethanol/NaOH). The effects of diazoxide on norepinephrine-contracted strips were recorded 10 min after addition of each concentration. The percentage changes in the height of norepinephrine-induced contractions were determined and the cumulative concentration–response curve of diazoxide-induced changes in the aortic strip contractions elicited by norepinephrine was constructed. In addition, the effects produced by pretreatment with either 400 µmol l−1 yohimbine (from a stock solution of 10 mmol l−1 in saline) or 100 µmol l−1 efaroxan (from a stock solution of 10 mmol l−1 in saline) for 15 min on this cumulative concentration–response curve of diazoxide were evaluated. Also, the alterations produced by pretreatment with 10 µmol l−1 glibenclamide (from a stock solution of 5 mmol l−1 in saline/ethanol/NaOH) alone or in combination with either 400 µmol l−1 yohimbine or 100 µmol l−1 efaroxan, for 15 min on the effects of different concentrations of diazoxide on the aortic strips

Determination of the plasma glucose and insulin levels

Chemicals The following chemicals were used: yohimbine hydrochloride (Miser Co., Egypt); glibenclamide (Hoechst Co., Egypt); efaroxan hydrochloride (Sigma Chemical Co., USA); streptozotocin (Sigma Chemical Co., USA); clonidine hydrochloride (Sigma Chemical Co., USA); diazoxide (Sigma Chemical Co., USA); D -glucose (El Naser Pharmaceutical and Chemical Co., Egypt) and pentobarbital sodium (Sigma Chemical Co., USA). All other chemicals were of analytical grade.

Statistical analysis of results The variability of results is expressed as mean ± SE. The significance of differences between mean values was determined by Student’s t-test.

RESULTS

Effect of yohimbine and efaroxan on oral glucose tolerance in non-diabetic control, type-I, and type-II diabetic rats Results presented in Figs 1 and 2 show that the oral administration of a glucose load of 2 g kg−1 to non-diabetic control rats produced a rapid increase in the plasma glucose and insulin levels, followed by a progressive decline until they nearly reached control values at the end of the 120 min period of investigation. In type-I diabetic rats, there was a marked intolerance to the oral glucose associated with a concomitant failure in induction of insulin secretion. In type-II diabetic rats, there was an intolerance to the oral glucose and a reduction in glucose-induced insulin secretion. Pretreatment of non-diabetic control rats with either 20 mg kg−1 yohimbine i.p. or 5 mg kg−1 efaroxan orally for 30 min significantly reduced the plasma glucose level elevated by the oral glucose load [Fig. 1(a)]. The marked elevation in plasma glucose level of type-I diabetic rats induced by oral administration of 2 g kg−1 glucose did not change significantly by the same pretreatment with either yohimbine or efaroxan [Fig. 1(b)]. Pretreatment of type-II diabetic rats with either 20 mg kg−1 yohimbine i.p. or 5 mg kg−1 efaroxan

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Fig. 1. The effect of 30 min pretreatment with either 20 mg kg−1 yohimbine (Yoh.) i.p. or 5 mg kg−1 efaroxan (Ef.) orally on oral glucose (Glu.) tolerance in non-diabetic control (a), type-I (b) and type-II (c) diabetic rats. The plasma glucose level was evaluated directly before and 15, 30, 60 and 120 min after administration of 2 g kg−1 Glu. orally. Values are means ± SE of six experiments. N, Glu.; , Yoh. + Glu.; , Ef. + Glu. ∗ P< 0.05; ∗∗ P< 0.01 vs corresponding Glu. values.

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orally for 30 min produced a significant reduction in the plasma glucose level elevated by the oral glucose load [Fig. 1(c)]. Pretreatment with either 20 mg kg−1 yohimbine i.p. or 5 mg kg−1 efaroxan orally for 30 min enhanced the elevation in the plasma insulin level induced in non-diabetic control rats by the oral administration of 2 g kg−1 glucose [Fig. 2(a)] and produced no change in plasma insulin level of type-I diabetic rats [Fig. 2(b)]. The elevation in the plasma insulin level induced in type-II diabetic rats by the oral glucose load was potentiated by the same pretreatment with either yohimbine or efaroxan [Fig. 2(c)].

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Fig. 2. The effect of 30 min pretreatment with either 20 mg kg−1 yohimbine (Yoh.) i.p. or 5 mg kg−1 efaroxan (Ef.) orally on oral glucose (Glu.) load-induced alterations in the plasma insulin level of non-diabetic control (a), type-I (b) and type-II (c) diabetic rats. The plasma insulin level was evaluated directly before and 15, 30, 60 and 120 min after administration of 2 g kg−1 Glu. orally. Values are means ± SE of six experiments. N, Glu.; , Yoh. + Glu.; , Ef. + Glu. ∗ P< 0.05; ∗∗ P< 0.01 vs corresponding Glu. values.

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Effect of yohimbine and efaroxan on plasma glucose and insulin levels of non-diabetic control, type-I and type-II diabetic rats Data listed in Fig. 3(a) illustrate that the i.p. injection of 20 mg kg−1 yohimbine or the oral administration of 5 mg kg−1 efaroxan to non-diabetic control rats reduced the plasma glucose level. This decrease was

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Fig. 3. Time course of changes in the plasma glucose level of nondiabetic control (a), type-I (b) and type-II (c) diabetic rats receiving either 20 mg kg−1 yohimbine (Yoh.) i.p. or 5 mg kg−1 efaroxan (Ef.) orally. Values are means ± SE of six experiments. , Yoh.; , Ef. ∗ P< 0.05; ∗∗ P< 0.01 vs zero-time values.

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observed after 60 and 120 min of drugs administration. The maximal percentages of reduction were 12 and 22% and occurred after 60 min of yohimbine and efaroxan administration, respectively. In type-I diabetic rats, yohimbine or efaroxan produced insignificant change in the plasma glucose level [Fig. 3(b)]. The i.p. injection of 20 mg kg−1 yohimbine or the oral administration of 5 mg kg−1 efaroxan to type-II diabetic rats produced a marked decrease in the plasma glucose level. This decrease was observed after 60 and 120 min of drugs administration. The maximal percentages of reduction were 20 and 31% and occurred after 60 min of yohimbine and efaroxan administration, respectively [Fig. 3(c)]. The plasma insulin level of non-diabetic control rats

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Fig. 4. Time course of changes in the plasma insulin level of nondiabetic control (a), type-I (b) and type-II (c) diabetic rats receiving either 20 mg kg−1 yohimbine (Yoh.) i.p. or 5 mg kg−1 efaroxan (Ef.) orally. Values are means ± SE of six experiments. , Yoh.; , Ef. ∗ P< 0.05; ∗∗ P< 0.01 vs zero-time values.

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increased significantly after 60 and 120 min of either i.p. injection of 20 mg kg−1 yohimbine or oral administration of 5 mg kg−1 efaroxan. The maximal percentages of increase were 31 and 43% and occurred after 60 min of yohimbine and efaroxan administration, respectively [Fig. 4(a)]. The same treatment of type-I diabetic rats with either yohimbine or efaroxan produced no change in the plasma insulin level over 120 min [Fig. 4(b)]. In typeII diabetic rats the plasma insulin level was increased after 60 and 120 min of either i.p. injection of 20 mg kg−1 yohimbine or oral administration of 5 mg kg−1 efaroxan. The maximal percentages of increase were 55 and 73% and occurred after 60 min of yohimbine and efaroxan administration, respectively [Fig. 4(c)].

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The subcutaneous injection of 300 µg clonidine to non-diabetic control and type-II diabetic rats produced an increase in the plasma glucose level. This effect was obvious after 30, 60 and 120 min of clonidine injection. In type-I diabetic rats clonidine produced no change in the plasma glucose level (Fig. 5). Pretreatment of non-diabetic control rats with either 20 mg kg−1 yohimbine i.p. or 5 mg kg−1 efaroxan orally for 30 min reduced the plasma glucose level elevated by the subcutaneous injection of 300 µg kg−1 clonidine [Fig. 5(a)]. The same pretreatment with either yohimbine or efaroxan did not change the plasma glucose level of type-I diabetic rats [Fig. 5(b)], but reduced the plasma glucose level of type-II diabetic rats elevated by the subcutaneous injection of 300 µg kg−1 clonidine [Fig. 5(c)]. In non-diabetic control and type-II diabetic rats, the plasma insulin level was decreased after 30, 60 and 120 min of subcutaneous injection of 300 µg kg−1 clonidine. The same treatment with clonidine produced no change in the plasma insulin level in type-I diabetic rats (Fig. 6). The reduction in the plasma insulin level induced by subcutaneous injection of 300 µg kg−1 clonidine was suppressed or reversed by 30 min pretreatment of nondiabetic control rats with either 20 mg kg−1 yohimbine i.p. or 5 mg kg−1 efaroxan orally [Fig. 6(a)]. The same pretreatment with either yohimbine or efaroxan produced no change in the plasma insulin level of type-I diabetic rats [Fig. 6(b)], but suppressed or reversed the reduction in the plasma insulin level induced by subcutaneous injection of 300 µg kg−1 clonidine into type-II diabetic rats [Fig. 6(c)]. Results presented in Fig. 7 illustrate that oral administration of 5 mg kg−1 glibenclamide to nondiabetic control and type-II diabetic rats produced a progressive reduction in the plasma glucose level. This effect started after 30 min of glibenclamide administration. Oral administration of 5 mg kg−1 glibenclamide to type-I diabetic rats decreased the plasma glucose level only after 120 min of its administration. The progressive reduction in the plasma glucose level induced by administration of 5 mg kg−1 glibenclamide orally to non-diabetic control rats was potentiated by 30 min pretreatment with either 20 mg kg−1 yohimbine i.p. or 5 mg kg−1 efaroxan orally [Fig. 7(a)]. The same pretreatment with either yohimbine or efaroxan produced no change in type-I diabetic rats [Fig. 7(b)], and potentiated the progressive reduction in the plasma glucose level induced by oral administration of 5 mg kg−1 glibenclamide in type-II diabetic rats [Fig. 7(c)]. The plasma insulin level of non-diabetic control and type-II but not of type-I diabetic rats was increased after 30, 60 and 120 min of glibenclamide administration (Fig. 8). kg−1

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Fig. 5. The effect of 30 min pretreatment with either 20 mg kg−1 yohimbine (Yoh.) i.p. or 5 mg kg−1 efaroxan (Ef.) orally on clonidine (Clo.)-induced alterations in the plasma glucose level of non-diabetic control (a), type-1 (b) and type-II (c) diabetic rats. The plasma glucose level was evaluated directly before and 15, 30, 60 and 120 min after subcutaneous injection of 300 µg kg−1 Clo. Values are means ± SE of six experiments. N, Clo.; , Yoh. + Clo.; , Ef. + Clo. ∗ P< 0.05; ∗∗ P< 0.01 vs corresponding Clo. values.

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The elevation in plasma insulin level of non-diabetic control rats induced by oral administration of 5 mg kg−1 glibenclamide was enhanced by pretreatment for 30 min with either 20 mg kg−1 yohimbine i.p. or 5 mg kg−1 efaroxan orally. There was a simple addition between yohimbine and glibenclamide and a synergism between

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Fig. 6. The effect of 30 min pretreatment with either 20 mg kg−1 yohimbine (Yoh.) i.p. or 5 mg kg−1 efaroxan (Ef.) orally on clonidine (Clo.)-induced alterations in the plasma insulin level of non-diabetic control (a), type-1 (b) and type-II (c) diabetic rats. The plasma insulin level was evaluated directly before and 15, 30, 60 and 120 min after subcutaneous injection of 300 µg kg−1 Clo. Values are means ± SE of six experiments. N, Clo.; , Yoh. + Clo.; , Ef. + Clo. ∗ P< 0.05; ∗∗ P< 0.01 vs corresponding Clo. values.

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Fig. 7. The effect of 30 min pretreatment with either 20 mg kg−1 yohimbine (Yoh.) i.p. or 5 mg kg−1 efaroxan (Ef.) orally on glibenclamide (Gl.)-induced alterations in the plasma glucose level of non-diabetic control (a), type-1 (b) and type-II (c) diabetic rats. The plasma glucose level was evaluated directly before and 15, 30, 60 and 120 min after oral administration of 5 mg kg−1 Gl. Values are means ± SE of six experiments. N, Gl.; , Yoh. + Gl.; , Ef. + Gl. ∗ P< 0.05; ∗∗ P< 0.01 vs corresponding Gl. values.

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efaroxan and glibenclamide [Fig. 8(a)]. Pretreatment of type-I diabetic rats similarly with either yohimbine or efaroxan produced no change in the plasma insulin level [Fig. 8(b)]. The same pretreatment with either yohimbine or efaroxan enhanced the stimulatory effect of glibenclamide on plasma insulin level of type-II diabetic rats. There was a simple addition between yohimbine and glibenclamide and a synergism between efaroxan and glibenclamide [Fig. 8(c)].

Data presented in Fig. 9 show that the i.p. injection of 30 mg kg−1 diazoxide into non-diabetic control and type-II diabetic rats produced a progressive increase in the plasma glucose level. This increase started after 30 min. The same dose of diazoxide produced no change in the plasma glucose level of type-I diabetic rats over 120 min.

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Fig. 8. The effect of 30 min pretreatment with either 20 mg kg−1 yohimbine (Yoh.) i.p. or 5 mg kg−1 efaroxan (Ef.) orally on glibenclamide (Gl.)-induced alterations in the plasma insulin level of non-diabetic control (a), type-I (b) and type-II (c) diabetic rats. The plasma insulin level was evaluated directly before and 15, 30, 60 and 120 min after oral administration of 5 mg kg−1 Gl. Values are means ± SE of six experiments. N, Gl.; , Yoh. + Gl.; , Ef. + Gl. ∗ P< 0.05; ∗∗ P< 0.01 vs corresponding Gl. values.

Fig. 9. The effect of 30 min pretreatment with either 20 mg kg−1 yohimbine (Yoh.) i.p. or 5 mg kg−1 efaroxan (Ef.) orally on diazoxide (Di.)-induced alterations in the plasma glucose level of non-diabetic control (a), type-I (b) and type-II (c) diabetic rats. The plasma glucose level was evaluated directly before and 15, 30, 60 and 120 min after i.p. injection of 30 mg kg−1 Di. Values are means ± SE of six experiments. N, Di.; , Yoh. + Di.; , Ef. + Di. ∗ P< 0.05; ∗∗ P< 0.01 vs corresponding Di. values.

Pretreatment of non-diabetic control rats with either 20 mg kg−1 yohimbine i.p. or 5 mg kg−1 efaroxan orally for 30 min reduced the plasma glucose level elevated by i.p. injection of 30 mg kg−1 diazoxide [Fig. 9(a)]. The same pretreatment with either yohimbine or efaroxan before diazoxide injection produced no change in the plasma glucose level in type-I diabetic rats [Fig. 9(b)], and reduced the plasma glucose level elevated by diazoxide in type-II diabetic rats [Fig. 9(c)]. The i.p. injection of 30 mg kg−1 diazoxide into nondiabetic control and type-II diabetic rats progressively

decreased the plasma insulin level. This effect started after 15 min. The same dose of diazoxide produced no change in the plasma insulin level of type-I diabetic rats over 120 min (Fig. 10). The reduction in the plasma insulin level induced by i.p. injection of 30 mg kg−1 diazoxide was antagonized and reversed by 30 min pretreatment of non-diabetic control rats with 20 mg kg−1 yohimbine i.p. and 5 mg kg−1 efaroxan orally, respectively [Fig. 10(a)]. Pretreatment of type-I diabetic rats similarly with either yohimbine or efaroxan before diazoxide injection

•

•

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405 100

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20 15

90 80 70 60 50 40 30 20 10 0

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60 75 Time (min)

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Fig. 11. The cumulative concentration–response curves of diazoxideinduced relaxation of isolated aortic strips contracted with 5 µmol l−1 norepinephrine in the absence ( ) and in the presence of 400 µmol l−1 yohimbine (); 10 µmol l−1 glibenclamide (N) and their combination ( ). Values are means ± SE of six experiments. ∗ P< 0.05; ∗∗ P< 0.01 vs diazoxide values.

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25 20 15 10 5 0

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0.1 Diazoxide (mmoll − 1)

100 90 80 70 60 50 40 30 20 10 0 0.01

25 20

0.1 Diazoxide (mmoll − 1)

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Fig. 12. The cumulative concentration–response curves of diazoxideinduced relaxation of isolated aortic strips contracted with 5 µmol l−1 norepinephrine in the absence ( ) and in the presence of 100 µmol l−1 efaroxan (); 10 µmol l−1 glibenclamide (N) and their combination ( ). Values are means ± SE of six experiments. ∗ P< 0.05; ∗∗ P< 0.01 vs diazoxide values.

◦

15

•

10 5 0

0

15

30

45

60 75 Time (min)

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Fig. 10. The effect of 30 min pretreatment with either 20 mg kg−1 yohimbine (Yoh.) i.p. or 5 mg kg−1 efaroxan (Ef.) orally on diazoxide (Di.)-induced alterations in the plasma insulin level of non-diabetic control (a), type-I (b) and type-II (c) diabetic rats. The plasma insulin level was evaluated directly before and 15, 30, 60 and 120 min after i.p. injection of 30 mg kg−1 Di. Values are means ± SE of six experiments. N, Di.; , Yoh. + Di.; , Ef. + Di. ∗ P< 0.05; ∗∗ P< 0.01 vs corresponding Di. values.

•

produced no change in the plasma insulin level [Fig. 10(b)]. The reduction in the plasma insulin level induced by i.p. injection of 30 mg kg−1 diazoxide in type-II diabetic rats was antagonized and reversed by 30 min pretreatment with 20 mg kg−1 yohimbine i.p. and 5 mg kg−1 efaroxan orally, respectively [Fig. 10(c)].

In vitro studies Figures 11 and 12 show the cumulative concentration– response curve of the effects of diazoxide on the contractions of the isolated aortic strips of rabbit elicited by 5 µmol l−1 norepinephrine. It is evident from the figures that the contractions of the aortic strips were inhibited in a concentration-dependent manner by diazoxide. When yohimbine in a concentration of 400 µmol l−1 was allowed to act for 15 min on isolated aortic strips contracted by norepinephrine, it produced no change in the relaxant effects of different concentrations of diazoxide. Addition of either 100 µmol l−1 efaroxan or 10 µmol l−1 glibenclamide to the fluid bathing isolated aortic strips contracted with 5 µmol l−1 norepinephrine, resulted after 15 min in inhibition of the relaxant effects of different concentrations of diazoxide on these strips. Results presented in the same figures also show that the concurrent addition of 100 µmol l−1 efaroxan and 10 µmol l−1 glibenclamide to the bathing fluid

406

completely inhibited the relaxant effects of different concentrations of diazoxide on the norepinephrinecontracted aortic strips. The concurrent addition of 400 µmol l−1 yohimbine and 10 µmol l−1 glibenclamide to the bathing fluid inhibited the relaxant effects of different concentrations of diazoxide on the norepinephrinecontracted aortic strips to an extent that did not differ significantly from that of glibenclamide alone. It is of interest that, in preliminary experiments, the concentration levels of yohimbine, efaroxan and glibenclamide used in this study did not produce by themselves any significant change on norepinephrinecontracted aortic strips.

DISCUSSION Previous studies demonstrated that neonatal streptozotocin injection induced, in adult rat, defects similar to those observed in mild type-II human diabetics: moderate increase in fasting blood glucose level, intolerance to oral glucose, hypoinsulinaemia and reduced glucoseinduced insulin secretion [19]. This model of type-II diabetes was compared to that of rats treated with streptozotocin when adult, which results in destruction of pancreatic beta-cells and is accepted as a model of type-I insulinopaenic diabetes [18]. Rats in this study responded to streptozotocin in a similar manner. Treatment of adult rats with streptozotocin produced a profound increase and decrease in the fasting blood glucose and insulin levels, respectively (i.e. a rat model of type-I diabetes). On the other hand, treatment of 1-day old rats with streptozotocin produced a relatively moderate increase and decrease in the fasting blood glucose and insulin levels, respectively (i.e. a rat model of type-II diabetes). It has been suggested that an increased sympathetic nervous system activity may be involved in the aetiology and pathogenesis of type-II diabetes. This hypothesis was based on the observation that both the stimulation of sympathetic nerves and the administration of selective alpha2 adrenoceptor agonists inhibited insulin secretion and induced hyperglycaemia [7]. On the other hand, abolition of the tonic adrenergic inhibition by alpha-2 adrenoceptor antagonists would lead to an antihyperglycaemic effect and improved glucose-induced insulin secretion [6]. On this basis, alpha-2 adrenoceptor antagonists may present a therapeutic interest and have been explored as potential therapeutic agents in diabetic patients [6, 9]. The data obtained in the present study indicate that pretreatment of non-diabetic control rats with selective alpha-2 adrenoceptor antagonists, yohimbine [6] or efaroxan [4] potentiated glucose-induced insulin release. Also, pretreatment of type-II but not of type-I diabetic rats with either yohimbine or efaroxan produced an improvement of oral glucose tolerance and potentiated glucose-induced insulin release. In studies of a similar nature, it has been found that efaroxan potentiated glucose-induced insulin

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secretion in rats [7]. Also, efaroxan potentiated glucoseinduced insulin secretion in the isolated pancreatic islets of rats [5, 21]. In addition, in several species including humans, SL 84.0418, a highly selective alpha-2 adrenoceptor antagonist, potentiated glucoseinduced insulin release and had an antihyperglycaemic effect [22]. This compound, was also found to improve glucose tolerance and to increase insulin secretion in a rat model of type-II diabetes [19, 22]. In light of these observations, the antihyperglycaemic effect of yohimbine and efaroxan obtained in this study in non-diabetic control and type-II diabetic rats could be explained in terms of potentiating glucose-induced insulin secretion. This explanation is easily confirmed since yohimbine and efaroxan neither improved oral glucose tolerance, nor increased insulin secretion in response to the glucose load in type-I diabetic rats. This possible insulinotropic effect of these agents was further supported in the present study after estimating their effects on plasma glucose and insulin levels of fasted non-diabetic control, type-I and type-II diabetic rats. Our results demonstrated that treatment of nondiabetic control and type-II diabetic rats with either yohimbine or efaroxan reduced the plasma glucose level and increased the plasma insulin level. Efaroxan was more effective than yohimbine in this respect. On the other hand, treatment of type-I diabetic rats with either yohimbine or efaroxan produced no change in both plasma glucose and insulin levels. These findings indicate that yohimbine and efaroxan decrease the plasma glucose level by stimulation of insulin secretion and have no extrapancreatic effects. Similar observations demonstrated that yohimbine induced an increase in plasma insulin level in fasted mice and rats [23, 24]. Also, efaroxan was found to increase the plasma insulin level and to reduce plasma glucose level in both conscious fasted and fed rats [7, 21]. Furthermore, efaroxan was found to stimulate insulin secretion from rat isolated pancreatic islets [8, 11]. In addition, it has been found that the release of insulin and hyperglycaemia induced by epinephrine in fasted male mice was markedly stimulated and inhibited, respectively, by phentolamine and yohimbine [23]. Also, Berridge et al. [7] found that efaroxan markedly antagonized the hyperglycaemic actions of epinephrine in rats and effectively potentiated the rise in insulin level produced by epinephrine. Furthermore, mammalian islets of Langerhans were found to contain substantial quantities of norepinephrine [25]. This agent is a powerful regulator of insulin release, the predominant response being an inhibition of secretion that results from activation of postsynaptic alpha-2 adrenoceptors [1]. Also, norepinephrine was shown to be a potent inhibitor of glucose-induced insulin release from rat pancreatic islets. This inhibition was antagonized by the alpha-2 adrenoceptor antagonist, yohimbine [25]. Efaroxan was found to antagonize the inhibitory effect of norepinephrine on insulin release [11].

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It is of interest that, in preliminary experiments, pretreatment with either yohimbine or efaroxan inhibited epinephrine and norepinephrine-induced hyperglycaemia, potentiated epinephrine-induced hyperinsulinaemia and prevented or reversed norepinephrineinduced hypoinsulinaemia in non-diabetic control and type-II diabetic rats. In type-I diabetic rats, epinephrine produced hyperglycaemia and no change in the plasma insulin level while norepinephrine produced no change in the plasma glucose and insulin levels. Pretreatment of animals with either yohimbine or efaroxan produced no change in the effects of epinephrine and norepinephrine. These results indicate that the hypoglycaemic effect of yohimbine and efaroxan in non-diabetic control and typeII diabetic rats is due to increase of insulin secretion by blocking of postsynaptic alpha-2 adrenoceptors located on the pancreatic beta-cells. Further evidence implicating the role of blocking of alpha-2 adrenoceptors of pancreatic beta-cells in the hypoglycaemic effect of yohimbine and efaroxan comes from investigating the effect of interaction of these drugs with clonidine on the plasma glucose and insulin levels. Our results demonstrated that clonidine increases the plasma glucose level and decreases the plasma insulin level in non-diabetic control and type-II but not in type-I diabetic rats. Pretreatment of animals with either yohimbine or efaroxan inhibited clonidine-induced hyperglycaemia and suppressed or reversed clonidineinduced hypoinsulinaemia. Clonidine, a selective alpha-2 agonist, induced its hyperglycaemic and hypoinsulinaemic effects by virtue of its activation of postsynaptic alpha-2 adrenoceptors located in pancreatic beta-cells [14, 23, 25, 26]. A number of studies have provided results which have cast doubt on the conclusion that the stimulatory effects of alpha-2 adrenoceptor antagonists on insulin secretion can be solely attributed to adrenoceptor blockade. Alpha-2 adrenoceptor antagonists that are imidazoline compounds (e.g. phentolamine and efaroxan) share the unusual property of eliciting a direct increase in insulin secretion in the absence of any adrenoceptor agonist [5, 10, 11]. It has been established that the primary determinant of this secretagogue activity is not alpha-2 antagonism per se, but rather the possession of an imidazoline ring within the molecule. It has been suggested that there are separate imidazoline binding sites of imidazoline receptors, which are not alpha-2 adrenoceptors, in islets. Thus, alpha-2 antagonists of the imidazoline family are able to stimulate insulin secretion on their own by binding to these sites [8, 14]. It has become evident from any studies that the gating of KATP channels in pancreatic beta-cells can be regulated by a variety of pharmacological agents. The most potent of these agents are the sulfonylureas which are used widely in the management of type-II diabetes mellitus [27]. The ability of sulfonylureas e.g. glibenclamide, to lower the plasma glucose concentration in diabetics appears mainly to be due to their ability to

407

stimulate insulin secretion. The ability of sulfonylureas to block the beta-cell KATP channels directly explains their stimulatory effect on insulin secretion [28]. On the other hand, diazoxide was found to inhibit insulin release by selectivity opening of KATP channels of pancreatic beta-cells, leading to hyperpolarization of the plasma membrane [29, 30]. In addition, it has been reported that an imidazolinebinding site might be involved in the control of KATP channels in beta-cells [15]. Also, it has been established that the activity of beta-cell KATP channels can also be controlled by certain imidazoline compounds [5, 16]. Many investigators [3, 5, 8, 11, 14, 22] suggested that the enhancement effect of certain imidazoline compounds (e.g. phentolamine and efaroxan) on insulin secretion results from depolarization of the beta-cell caused by closure of KATP channels. It was suggested that these effects do not result from interaction of the compounds with alpha-2 adrenoceptors, since only certain alpha-2 adrenoceptor antagonists are effective as KATP blockers and insulin secretagogues. Moreover, some other imidazoline compounds stimulate insulin secretion but do not interact with alpha-2 adrenoceptors. Furthermore, some investigators have reported that the non-imidazoline alpha-2 adrenoceptor antagonist, yohimbine, inhibits KATP channels in pancreatic betacells [31]. In view of these considerations, the role of KATP channels in the hypoglycaemic activity of yohimbine and efaroxan was evaluated in the present work by studying the interaction between each of these drugs and glibenclamide or diazoxide. The data obtained in this study indicate that administration of glibenclamide to non-diabetic control and type-II diabetic rats produced a marked reduction in the plasma glucose level and a marked increase in the plasma insulin level. In type-I diabetic rats, glibenclamide produced a slight decrease in the plasma glucose level only at the end of the investigation period, but there was no change in the plasma insulin level. Pretreatment of animals with either yohimbine or efaroxan enhanced the hypoglycaemic and insulinotropic effects of glibenclamide in non-diabetic control and type-II diabetic rats, but produced no change in glibenclamide effects in type-I diabetic rats. On the other hand, diazoxide produced an increase in the plasma glucose level and a decrease in the plasma insulin level in non-diabetic control and type-II but not in type-I diabetic rats. The effect of diazoxide on the plasma glucose level was inhibited by pretreatment of these animals with either yohimbine or efaroxan, while its effect on the plasma insulin level was antagonized and reversed by pretreatment with yohimbine and efaroxan, respectively. However, our results indicate that the combination of glibenclamide and efaroxan led to a synergistic increase in insulin secretion, while the combination of glibenclamide and yohimbine led to an additive increase. On the other hand, efaroxan reversed the hypoinsulinaemic effect of diazoxide, while yohimbine

408

antagonized it. In view of the previous considerations, these findings are in favour of the possibility that efaroxan blocked KATP channels in the pancreatic beta-cell membrane, while yohimbine did not. The first evidence that KATP channels exist in smooth muscle came from electrophysiological measurements of these channels in smooth muscle and the observation that the vasodilating actions of diazoxide were inhibited by glibenclamide [32]. Then the evidence has accumulated that KATP channels in vascular smooth muscle are the target of a variety of vasodilating stimuli [33–35]. The demonstration of KATP channels in arterial smooth muscle [32] argues for the presence of sulfonylurea binding sites in arterial smooth muscle [36]. In endotheliumdenuded rings from rat aorta, a high affinity component of [3 H] glibenclamide binding mediates the block of KATP channels by sulfonylureas in rat aorta,hence, it represents the sulfonylurea receptor in this vessel. The pharmacological properties of this binding site resemble those of the binding site for the openers of KATP channels [37]. It has been found that glibenclamide antagonized, in a concentration-dependent manner, the vasorelaxant effects of the KATP channel openers, cromakalim, minoxidil and diazoxide on isolated rabbit aorta contracted with angiotensin II, norepinephrine or methoxamine [38]. Furthermore, diazoxide, a potent activator of KATP channels in pancreatic beta-cells, fails to discriminate between vascular smooth muscle and beta-cell KATP channels [39, 40]. It has also been reported that imidazoline binding sites play a role in inhibition of KATP channels in vascular smooth muscle [41, 42]. In addition, it has been demonstrated that rabbit aorta and rat aorta contain alpha-1 adrenoceptors [43–45]. Prazosin was found to be more potent than phentolamine, and phentolamine was found to be more potent than yohimbine, in antagonism of norepinephrine or phenylephrine-induced contraction of rabbit aortic strips [46]. In the present study, in vitro experiments using isolated rabbit aortic strips illustrate further the role of KATP channels in the hypoglycaemic effect of yohimbine and efaroxan. Our results demonstrate that the bath application of efaroxan or glibenclamide inhibited the relaxant effects of different concentrations of diazoxide on the isolated norepinephrine-contracted aortic strips, while the application of yohimbine produced insignificant changes. The combination of glibenclamide and efaroxan led to complete inhibition of the relaxant effects of different concentrations of diazoxide, while the combination of glibenclamide and yohimbine did not produce such an effect. Taken together, our results suggest that the alpha-2 adrenoceptor antagonists i.e. yohimbine and efaroxan, decrease the plasma glucose level in control and type-II diabetics. This effect is mediated by releasing insulin. The insulinotropic effect of yohimbine, a nonimidazoline compound, is due to antagonism at postsynaptic alpha-2 adrenoceptors located on pancreatic beta-

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cells. The insulinotropic effect of efaroxan, an imidazoline compound, may be due to the dual actions of antagonism at postsynaptic alpha-2 adrenoceptors and blockade of KATP channels in pancreatic beta-cell membranes.

REFERENCES 1. Wang SY, Pilkey DT. Identification in islets of Langerhans of a new rat alpha-2 adrenoceptor. Diabetes 1994; 43: 127–36. 2. Chan SLF, Brown CA, Morgan NG. Stimulation of insulin secretion by imidazoline alpha-2 adrenoceptor antagonist efaroxan is mediated by a novel stereoselective binding site. Eur J Pharmacol 1993; 230: 375–8. 3. Morgan NG, Chan SLF, Brown CA, Tsoli E. Characterization of the imidazoline binding site involved in regulation of insulin secretion. Ann NY Acad Sci 1995; 763: 361–73. 4. Berridge TL, Collins PD, Roach AG. Effect of efaroxan, an alpha2 adrenoceptor antagonist, on insulin release in conscious rats. Br J Pharmacol 1989; 96: 288P–93P. 5. Chan SLF, Morgan NG, Dunne MJ, Stillings MR. The alpha2 adrenoceptor antagonist efaroxan modulates K+ ATP channels in insulin-secreting cells. Eur J Pharmacol 1991; 204: 41–5. 6. Broadstone VL, Pfeifer MA, Stagner JI, Samols E. Alphaadrenergic blockade improves glucose-potentiated insulin secretion in non-insulin-dependent diabetes mellitus. Diabetes 1987; 36: 932–6. 7. Berridge TL, Doxey JC, Roach AG. Comparison of the effects of efaroxan and glibenclamide on plasma glucose and insulin levels in rats. Eur J Pharmacol 1992; 213: 213–8. 8. Brown CA, Chan SLF, Stilling MR, Smith SA, Morgan NG. Antagonism of the stimulatory effects of efaroxan and glibenclamide in rat pancreatic islets by the imidazoline RX 801080. Br J Pharmacol 1993; 110: 1017–22. 9. Berlin I, Rosenzweig P, Fuseau E, Molinier P. Effect of a new alpha-2 adrenoceptor antagonist (SL 84.0418) on C-peptide and insulin responses to glucose infusion in healthy subjects. Diabetologia 1991; 34: 107–10. 10. Schulz A, Hasselblatt A. Phentolamine: a deceptive tool to investigate sympathetic nervous control of insulin release. Naunyn Schmiedebergs Arch Pharmacol 1988; 337: 637–40. 11. Chan SLF, Morgan NG. Stimulation of insulin secretion by efaroxan may involve interaction with potassium channels. Eur J Pharmacol 1990; 176: 97–101. 12. Ashcroft FM, Williams B, Smith PA, Fewtrell CMS. Ion channels involved in the regulation of nutrient-stimulated insulin secretion. In: Nutrient regulation of insulin secretion. Flatt PR, ed. London: Portland Press, 1992: 193–212. 13. Zhang Y, Warren-Perry M, Sakura H, Turner R. No evidence for mutations in a putative beta-cell. ATP-sensitive K+ channel subunit in MODY, NIDDM or GDM. Diabetes 1995; 44: 597–600. 14. Schulz A, Hasselblatt A. Dual action of clonidine on insulin release: suppression but stimulation when alpha-2 adrenoceptors are blocked. Naunyn Schmiedebergs Arch Pharmacol 1989; 340: 712–4. 15. Plant T, Jonas JC, Henquin JC. Clonidine inhibits ATP-sensitive K+ channels in mouse pancreatic beta-cells. Br J Pharmacol 1991; 104: 385–90. 16. Chapman JC, McClenaghan NH, Cosgrove KE, Hashmi MN, Shepherd RM, Dunne MJ. ATP-sensitive potassium channels and efaroxan-induced insulin release in the electrofusion-derived brain-BD 11 beta-cell line. Diabetes 1999; 48: 2349–57. 17. Morgan NG, Chan SLF, Mourtada M, Ramsden CA. Imidazoline and pancreatic hormone secretion. Ann NY Acad Sci 1999; 881: 217–28. 18. Burcelin R, Printz RL, Kande J, Assan R, Granner DK, Girard J. Regulation of glucose transporter and hexokinase-II expression in tissues of diabetic rats. Am J Physiol 1993; 265: E392–401. 19. Angel I, Burcelin R, Girard J, Salomon Z. Normalization of insulin secretion by selective alpha-2 adrenoceptor antagonist receptors

Pharmacological Research, Vol. 44, No. 5, 2001

20. 21.

22.

23.

24.

25.

26.

27.

28. 29.

30.

31.

32.

33.

GLUT-4 glucose transporter expression in adipose tissue of typeII diabetic rats. Endocrinology 1996; 137: 2022–7. Crofford OB, Davis CK. Evaluation of a new technique of multiple serial sampling. Metabolism 1965; 14: 271–5. Mourtada M, Brown AC, Smith SA, Chan LF. Interaction between imidazoline compounds and sulphonylureas in the regulation of insulin secretion. Br J Pharmacol 1997; 121: 799–805. Berlin I, Rosenzweig P, Fuseau Chalon ELS, Landault C, Cesselin F, Blacker C, Puech AJ. Reduction of hyperglycemia after oral glucose load by the new alpha-2 adrenergic receptor antagonist SL 84.0418 in healthy subjects. Clin Pharmacol Ther 1994; 55: 338–45. Nakadate T, Nakaki T, Muraki T, Kato R. Adrenergic regulation of blood glucose levels: possible involvement of postsynaptic alpha2 type adrenoceptors regulating insulin release. J Pharmacol Exp Ther 1980; 215: 226–30. Ahren B, Jarhalt J, Lundquist I. Enhancement of insulin secretion during selective blockade of alpha-1 and alpha-2 adrenoceptors in rat. Effect of somatostatin. Acta Physiol Scand 1982; 115: 257–260. Nakaki T, Nakadate T, Kato R. Alpha-2-adrenoceptors modulating insulin release from isolated pancreatic islets. Naunyn Schmiedebergs Arch Pharmacol 1980; 313: 151–6. Metz SA, Halter B, Robertson RP. Induction of defective insulin secretion and impaired glucose tolerance by clonidine. Diabetes 1978; 27: 554–62. Nelson DA, Aguilar-Bryan L, Raef H, Boyd A. Molecular mechanisms of sulphonylurea in the pancreatic beta-cells. In: Nutrient regulation of insulin secretion. Flatt PR, ed. London: Portland Press, 1992: 319–40. Satin LS. New mechanisms for sulphonylurea control of insulin secretion. Endocrine 1996; 4: 191–8. Dunne MJ. Protein phosphorylation is required for diazoxide to open ATP-sensitive K+ channels in insulin (RINm5F) secreting cells. FEBS Lett 1989; 250: 262–6. Sato Y, Aizawa T, Komatsu M, Okada N, Yamada T. Dual functional role of membrane depolarization Ca++ influx in rat pancreatic beta-cell. Diabetes 1992; 41: 438–43. Plant T, Henquin JC. Phentolamine and yohimbine inhibit ATPsensitive K+ channels in mouse pancreatic beta-cells. Br J Pharmacol 1990; 101: 115–20. Standen NB, Quayle JM, Davies NW, Nelson MT. Hyperpolarizing vasodilators activate ATP-sensitive K+ channels in arterial smooth muscle. Science 1989; 245: 177–80. Nelson MT, Quayle JM. Physiological roles and properties of potassium channels in arterial smooth muscle. Am J Physiol 1995;

409 268: C799–822. 34. Yokoshiki H, Sunagawa W, Seki T, Sperelakis N. ATP-sensitive K+ channels in pancreatic, cardiac, and vascular smooth muscle cells. Am J Physiol 1998; 274: C25–37. 35. Fujita A, Kurachi Y. Molecular aspects of ATP-sensitive K+ channel in the cardiovascular system and K+ channel openers. Pharmacol Ther 2000; 85: 39–53. 36. Kovacs RJ, Nelson MT. ATP-sensitive K+ channels from aortic smooth muscle incorporated into planar lipid bilayers. Am J Physiol 1991; 261: H1604–9. 37. Loeffler C, Quast U. Pharmacological characterization of the sulphonylurea receptor in rat isolated aorta. Br J Pharmacol 1997; 120: 476–80. 38. Winquist RJ, Heaney LA, Wallace AA, Kaczarowski GJ. Glyburide blocks the relaxation response to BRL 3415 (cromakalim), minoxidil, sulfate and diazoxide in vascular smooth muscle. J Pharmacol Exp Ther 1989; 248: 149–56. 39. Antoine M, Berkenboom G, Herchuelz A, Lebrun P. Mechanical and ionic response of rat aorta to diazoxide. Eur J Pharmacol 1992; 216: 299–306. 40. De Tullio P, Pirotte B, Antoine M, Delarge J. 3 and 4 substituted 4H-pyrido thiadiazine dioxides as potassium channel openers. Synthesis, pharmacological evaluation and structure activity relationships. J Med Chem 1996; 47: 4525–9. 41. McPherson GA, Angus JA. Phentolamine and structurally related compounds selectively antagonize the vascular actions of the K+ channel opener, Cromakalim. Br J Pharmacol 1989; 97: 941–9. 42. Regunathan S, Youngson C, Wang H, Reis DJ. Imidazoline receptors in vascular smooth muscle and endothelial cells. Ann NY Acad Sci 1995; 763: 580–90. 43. Ruffolo RR, Waddel JE, Yaden EL. Postsynaptic alpha adrenoceptor subtypes. Differentiated by yohimbine in tissues from rat. Existence of alpha-2 adrenergic receptor in rat aorta. J Pharmacol Exp Ther 1981; 217: 235–40. 44. Piascik MT, Sparks MS, Pruitt TA. Interaction of imidazolines with alkylation-sensitive and resistant alpha-1 adrenoceptor subtypes. J Pharmacol Exp Ther 1991; 258: 158–65. 45. Muramatsu I, Murata S, Isaka M, Piao HL, Miyamoto S, Oshita M, Watanabe Y, Taniguchi T. Alpha-1 adrenoceptor subtypes and two receptor systems in vascular tissues. Life Sci 1998; 62: 1461–5. 46. Takeuchi K, Akatsuka K, Kasuya Y. Pharmacological studies of imidazoline derivatives. II. Agonistic and antagonistic actions on alpha-adrenoceptors in various smooth muscle preparations. J Pharmacobiodyn 1986; 9: 385–94.