British Journal of Pharmacology and Toxicology Research Vinpocetine

British Journal of Pharmacology and Toxicology

Research

Vinpocetine, sildenafil have significant insulinotropic action from isolated islets and antagonize the action of gliclazide.

A. S. El-Deeb*1; N. I. Eid1; M. E. El-Sayed1

1Pharmacology & Toxicology Department, Faculty of Pharmacy, Cairo University,

11562, Egypt.

Abstract

Cyclic nucleotides play a pivotal role in the glucose-induced insulin release; however, the effect of phosphodiesterase inhibitors (PDEIs) is controversial. Hence, the aim of the present study was to construct a correlation between cGMP level and insulin release. To fulfill this aim, isolated pancreatic islets were exposed to different concentrations of vinpocetine, sildenafil, or the standard drug gliclazide alone or in combination. The study showed that the high concentrations (40, 80 µmol/l) of either vinpocetine or sildenafil and gliclazide (10, 20, 40, 80 µmol/l) raised the insulin/cGMP release in the presence of basal (3mmol/l) or stimulatory (16.7 mmol/l) glucose levels. However, the low concentrations (10, 20 µmol/l) of vinpocetine or sildenafil elevated the insulin release in the presence of stimulatory glucose level. At the two glucose levels, all combinations increased insulin secretion, except for the 10µmol/l concentration of both vinpocetine and sildenafil, which elevated insulin secretion in the supra-physiological level only. The same pattern was detected in regard to the effect on cGMP. The study also showed a highly significant correlation between both insulin and cGMP levels.

In conclusion, both PDIs have insulinotropic effects and increase the release of cGMP,

but the effect of vinpocetine superseded that of sildenafil. Moreover, combining both

PDEIs antagonize the insulinotropic effect of each other or of gliclazide, possibly due to

the negative feedback mechanism of cGMP that holds it from augmenting insulin

secretion at certain level.

Keywords: cGMP, gliclazide, insulin,pancreatic islets, sildenafil, vinpocetine.

Introduction:

Cyclic nucleotides, viz., cGMP and cAMP were reported to play a role in the augmentation of insulin secretion (Smukler et al., 2002; Ishikawa et al., 2003; Nystrom et al., 2012) and were found to be regulated according to the activity of the phosphodiesterase (PDE) enzyme (Domek-Lopacinska et al., 2005).

PDE was further subclassified into 11 subtypes, with the PDE3B (PDE3) being the most predominant isoform involved in regulation of insulin release beside the contribution of other subtypes, such as PDE1, PDE4, PDE10 (Pyne et al., 2003; Waddleton et al., 2008;

Heimann et al., 2010).

Ample of evidence has pointed to the antidiabetic effect of the phosphodiesterase inhibitors (PDEIs); however, these results were controversial (Pour et al, 2007).

Vinpocetine is a classic inhibitor of PDE1 that elevates the intracellular level of cGMP and cAMP (Medina, 2011) and is able to block voltage-dependent Na+ (Molnar et al., 1995) and Ca2+(Sitges et al., 2007)channels. Besides, Vinpocetine possessed an antioxidant effect, which is capable of attenuating oxidative stress (Zaitone et al., 2012;Zaki et al., 2013).

Another PDI that possesses also an antioxidant activity (Rodrigues et al., 2013) is

Sildenafil. The drug acts as a selective inhibitor of the cGMP-specific phosphodiesterase type V (PDE5), which increases intracellular cGMP level (Cheitlin et al., 1999).

Based on the above data our study aimed to test the previously mentioned PDIs at different concentrations, alone or in combination, on the release of both insulin and cGMP using the isolated pancreatic model as compared to Gliclazide, the standard antidiabetic drug used. Additionally, we tested the effect of either PDI on the Gliclazide-induced insulin/cGMP release and verify the possible correlation between cGMP level and insulin release.

Material and methods

Animals: Adult male wistar albino rats weighing 250 – 300 g were used. They were

obtained from animal house of faculty of pharmacy of Cairo University, Cairo, Egypt.

The animals were kept in plastic cages and allowed to accommodate for one week before

being subjected to experimentation. They were fed purina-chew, water was allowed ad-

libitum. The study was conducted in accordance with ethical procedures and policies

approved by the ethics of committee of faculty of pharmacy, Cairo University.

Drugs and chemicals:Glcl powder supplied as a gift from Memphis Pharmaceutical and

Chemical Industries, Egypt. Vinp and Sild powders were supplied as a gift from Global

Napi pharmaceuticals, Egypt. Rat insulin ELISA Kit was purchased from ALPCO, USA.

Any another analytical chemicals are of equal quality or A.R. quality.

Isolation and incubation of pancreatic islets: Pancreatic islets were isolated following

collagenase digestion technique according to the method of Lacy and

Kostianovsky(1967). The islets were pre-incubated into Wassermann tube containing

3 fresh Krebs-Ringer-HEPES (KRH) solution and incubated at 37˚C for 30 min in a shaking water bath for adaptaion (Lacy et al., 1968). Batches of 5 islets were picked up and incubated in small tubes, each containing 1 ml KRH buffer supplemented with 0.5% bovine serum albumin, glucose 3 or 16.7 mmol/l and the test drugs.

Experimental design: Isolatedpancreatic islets were divided into 50 groups. 24 groups incubated in media supplemented with glucose 3 mmol/l where one of these groups was considered as normal control. On the other hand, the another 24 groups incubated within media supplemented with glucose 16.7 mmol/l and one of these groups was considered as normal control. Each group consists of 6 Wasserman tubes, each containing batch of 5 islets with 0.5% bovine serum albumin. The drugs were added individually in concentrations 10, 20, 40, 80 µmol/l. while the drugs were combined together with following concentration patterns (10 + 10, 20 + 20, 40 + 40, 80 + 80 µmol/l). The tubes were covered and incubated at 37˚C in a shaking water bath for 1 h, then the tubes were transferred into ice-bath, mixed with vortex mixer and aliquots 0.5 ml were taken and kept frozen at -20˚C for determination of biochemical parameters.

Determination of biochemical parameters: Insulin and cGMP levels were measured by enzyme-linked immunosorbent assay (ELISA) which is based on the sandwich principle using kits (ALPCO, USA) and (Glory Science Co. Ltd, China), respectively.

Statistical analysis: Data were expressed as mean ± S.E.M. Statistical analysis was carried out by 2 ways repeated measures ANOVA followed by post-test Newmans-Keuls multiple comparison test for comparisons of means of different groups using Graph Pad

Prism 5 Program. Drug interaction were analyzed by two way ANOVA factorial design

4 using Microsoft Excel computer program.For all statistical tests, the level of significance was at P < 0.05. Graphical representations were designed by Graph Pad Prism 5 program.

Results

Effects of different concentrations of gliclazide, vinpocetine or sildenafil alone on insulin secretion in presence of glucose (3 mmol/l) from isolated pancreatic islets of rats: Gliclazide (10, 20, 40, 80 µmol/l), vinpocetine (40, 80 µmol/l), sildenafil (40, 80

µmol/l) significantly raised basal insulin secretion to 202.19, 239.68, 266.04, 321.8,

147.29, 237.92, 127.63, 147.72 % respectively compared to control value in a concentration-dependent manner. Gliclazide (10, 20, 40, 80 µmol/l) significantly raised basal insulin secretion from isolated pancreatic islets compared to vinpocetine (10, 20,

40, 80 µmol/l) and sildenafil (10, 20, 40, 80 µmol/l). on the other hand, vinpocetine (80

µmol/l) showed only significant increase of basal insulin secretion compared to sildenafil

(80 µmol/l) (Tab. 1).

Effects of different concentrations of combined treatments of gliclazide and vinpocetine or sildenafil as well as vinpocetine and sildenafil on insulin secretion in presence of glucose (3 mmol/l) from isolated pancreatic islets of rats: Addition of gliclazide and sildenafil (10 + 10, 20 + 20, 40 + 40, 80 + 80 µmol/l), gliclazide and vinpocetine (10 + 10, 20 + 20, 40 + 40, 80 + 80 µmol/l), vinpocetine and sildenafil (20 +

20, 40 + 40, 80 + 80 µmol/l) significantly increased basal insulin secretion from isolated pancreatic islets to 211.57, 233.42, 270.48, 339.2, 220.94, 269.14, 284.3, 360.63, 135.67,

167.8, 261.11 % respectively compared to control value in a concentration-dependent manner. Incubation of islets with gliclazide and sildenafil (10 + 10, 20 + 20, 40 + 40, 80

+ 80 µmol/l), gliclazide and vinpocetine (10 + 10, 20 + 20, 40 + 40, 80 + 80 µmol/l) significantly raised basal insulin secretion compared to sildenafil and vinpocetine (Fig.

2).

Effects of different concentrations of gliclazide, vinpocetine or sildenafil alone on insulin secretion in presence of glucose (16.7 mmol/l) from isolated pancreatic islets of rats: Gliclazide (10, 20, 40, 80 µmol/l), vinpocetine (10, 20, 40, 80 µmol/l), sildenafil

(10, 20, 40, 80 µmol/l) significantly elevated glucose (16.7 mmol/l) stimulated insulin secretion to 302.83, 326.19, 372.92, 409.15, 228.45, 242.11, 252.16, 275.16, 156.10,

192.44, 209.02, 248.65 % respectively compared to control value in a concentration- dependent manner. Gliclazide (10, 20, 40, 80 µmol/l) raised significantly stimulated insulin secretion from isolated pancreatic islets compared to vinpocetine (10, 20, 40, 80

µmol/l) and sildenafil (10, 20, 40, 80 µmol/l). However, vinpocetine (10, 20, 40, 80

µmol/l) showed significant increase of glucose (16.7 mmol/l) stimulatory insulin secretion compared to sildenafil (10, 20, 40, 80 µmol/l) (Fig. 3).

Effects of different concentrations of combined treatments of gliclazide and vinpocetine or sildenafil as well as vinpocetine and sildenafil on insulin secretion in presence of glucose (16.7 mmol/l) from isolated pancreatic islets of rats: Gliclazide and sildenafil (10 + 10, 20 + 20, 40 + 40, 80 + 80 µmol/l), gliclazide and vinpocetine (10

+ 10, 20 + 20, 40 + 40, 80 + 80 µmol/l), vinpocetine and sildenafil (10 +10, 20 + 20, 40

+ 40, 80 + 80 µmol/l) significantly raised glucose (16.7 mmol/l) stimulatory insulin secretion to 274.84, 323.03, 344.48, 367.5, 274.84, 323.03, 344.48, 378.4, 253.28,

280.14, 294.48, 311.08 % compared to control respectively in a concentration-dependent

6 manner. Combination of the smallest concentrations of gliclazide and vinpocetine (10 +

10 µmol/l) showed significant effect on stimulated insulin secretion compared to vinpocetine and sildenafil (10 + 10 µmol/l) or gliclazide and sildenafil (10 + 10 µmol/l)

(Tab. 4).

Effects of different concentrations of gliclazide, vinpocetine or sildenafil alone on cGMP level in presence of glucose (3 mmol/l) from isolated pancreatic islets of rats:

Incubation of gliclazide (10, 20, 40, 80 µmol/l), vinpocetine (20, 40, 80 µmol/l), sildenafil (40, 80 µmol/l) significantly increased basal cGMP level from isolated pancreatic islets to 176.34, 246.67, 279.56, 362.36, 154.2, 171.4, 229.03, 151.61, 188.17

% respectively compared to control value in a concentration-dependent manner.

Gliclazide (10, 20, 40, 80 µmol/l) significantly raised non-stimulated cGMP level from isolated pancreatic islets compared to vinpocetine and sildenafil of the same concentrations. Vinpocetine (80 µmol/l) showed significant increase of basal cGMP level compared to sildenafil (80 µmol/l) (Tab. 5).

Effects of different concentrations of combined treatments of gliclazide and vinpocetine or sildenafil as well as vinpocetine and sildenafil on cGMP level in presence of glucose (3 mmol/l) from isolated pancreatic islets of rats: Gliclazide and sildenafil (10 + 10, 20 + 20, 40 + 40, 80 + 80 µmol/l), gliclazide and vinpocetine (10 +

10, 20 + 20, 40 + 40, 80 + 80 µmol/l), vinpocetine and sildenafil (10 + 10, 20 + 20, 40 +

40, 80 + 80 µmol/l) significantly elevated cGMP level to 185.6, 255.91, 304.3, 379.56,

207.95, 297.2, 306.88, 418.7, 164.94, 186.45, 225.8, 250.96 % compared to control value in a concentration-dependent manner. Incubation of islets with gliclazide and

7 sildenafil (20 + 20, 40 + 40, 80 + 80 µmol/l) or gliclazide and vinpocetine (20 + 20, 40 +

40, 80 + 80 µmol/l) significantly raised basal cGMP level compared to sildenafil and vinpocetine at the same concentrations (Fig. 6).

Effects of different concentrations of gliclazide, vinpocetine or sildenafil alone on cGMP level in presence of glucose (16.7 mmol/l) from isolated pancreatic islets of rats: Gliclazide (10, 20, 40, 80 µmol/l), vinpocetine (20, 40, 80 µmol/l), sildenafil (40,

80 µmol/l) significantly elevated stimulated cGMP level to 135.01, 166.7, 209.15,

243.15, 149.27, 190.46, 232.54, 135.51, 168.31 % respectively compared to control value in a concentration-dependent manner. Although gliclazide (10, 20, 40, 80 µmol/l) raised significantly stimulated cGMP level from sildenafil, yet it didn’t significantly change it from vinpocetine at the same concentrations. However, vinpocetine (20, 40, 80 µmol/l) showed significant increase of cGMP level compared to sildenafil of the same concentrations (Fig. 7).

Effects of different concentrations of combined treatments of gliclazide and vinpocetine or sildenafil as well as vinpocetine and sildenafil on cGMP level in presence of glucose (16.7 mmol/l) from isolated pancreatic islets of rats: Combination of gliclazide and sildenafil (10 + 10, 20 + 20, 40 + 40, 80 + 80 µmol/l), gliclazide and vinpocetine (10 + 10, 20 + 20, 40 + 40, 80 + 80 µmol/l), vinpocetine and sildenafil (10

+10, 20 + 20, 40 + 40, 80 + 80 µmol/l) significantly increased glucose (16.7 mmol/l) stimulatory cGMP level from isolated pancreatic islets to 219.61, 253.39, 276.35, 318.37,

247.64, 303.02, 325.23, 358.84, 212.92, 210.88, 225.92, 250.98 % respectively compared

8 to control value in a concentration-dependent manner. Incubation of islets with gliclazide and sildenafil (20 + 20, 40 + 40, 80 + 80 µmol/l) or with gliclazide and vinpocetine (20 +

20, 40 + 40, 80 + 80 µmol/l) raised stimulated cGMP level significantly compared to sildenafil and vinpocetine of the same doses (Fig. 8).

Discussion

Cyclic AMP is an important physiological amplifier of glucose-induced insulin secretion

(Pyne, 2003).Glucose is the main stimulators of insulin release. Glucose metabolism in the β-cell elevates the cytosolic ATP/ADP ratio and closes ATP-sensitive K+-channel

2+ (KATP) resulting in β-cell depolarization, Ca influx through voltage dependent calcium channel (VDCC) and Ca2+ dependent exocytosis of insulin (Leech et al., 2011).

At glucose concentration (< 3 mM), the β-cell is electrically silent with a resting membrane potential (-70 mV). Increasing external glucose produces a slow depolarization till it reaches physiological concentration (> 7 mM), which elicits insulin release since the depolarization reaches the threshold potential ( -50 mV). When the glucose reaches (> 16mM), the duration of bursts reaches plateau where β-cell shows continuous action potentials at glucose concentration (Ashcroft and Rorsman, 1989).

In the present work, gliclazide increased insulin secretion from isolated pancreatic islets in concentration-dependent manner either in presence of basal or stimulatory glucose levels.

These results are in agreement with that recorded by (Del et al., 2009; Gregorio et al.,

1992; Lawrence et al., 2001).

In the current study, vinpocetine increased insulin secretion from isolated pancreatic islets in presence of supraphysiologlical glucose level in a concentration-dependent manner while it enhanced insulin secretion in presence of basal glucose level at higher concentrations namely; 40, 80 µmol/l.

Similarly, Han et al(1999) demonstrated that 8-methoxymethyl-isobutylmethylxanthine

(8MM-IBMX), a selective PDE1 inhibitor increased insulin secretion from pancreatic islets. Also, Heimann et al (2010), proved the presence of PDE1s in human and rat pancreatic islets and their inhibition potentiate glucose-stimulated insulin secretion.

Also, sildenafil significantly increased insulin secretion in presence of either basal or supra-physiological glucose concentration from isolated rat pancreatic islets.

These results are in accordance with that recorded by El-Deeb et al (2014), who demonstrated the insulinotropic effect of sildenafil on diabetic rats in vivo.

In the present work, vinpocetine significantly increased insulin secretion in comparison to sildenafil. These results are in accordance with that recorded by Shafiee-Nick et al (1995) who found that zaprinast (PDE1/PDE5 inhibitor) did not modify glucose –induced insulin release from pancreatic islets. This is rationalized due to the potent inhibition of zaprinast to PDE5 compared with PDE1 as rationalized by Ahmad et al (2000). This indicating the importance of PDE1 in insulin regulation in comparison to PDE5.

In the present work, gliclazide increased cGMP release from isolated pancreatic islets in presence of either basal or stimulatory glucose concentration.

These findings are in accordance with that observed by Grill et al.(1978) who stated that sulphonylurea exerts a major part of their action through facilitating glucose stimulated

10 adenylcyclase in the β-cell. This stimulation increases cAMP content which in turn increases insulin release.

Vinpocetine and sildenafil increased cGMP release from isolated pancreatic islets in presence of either basal or stimulatory glucose concentration.

(Ahn et al., 1989; Giachini et al., 2011; Hagiwara et al., 1984; Medina, 2011) et al recorded that vinpocetine increased cGMP through selective inhibition of Ca2+-dependent phosphodiesterase 1. On the other hand, (Boolell et al., 1996; Giachini et al., 2011;

Milani et al., 2005; Puzzo et al., 2009) demonstrated that the increased cGMP by sildenafil is due to selective inhibition of PDE5.

In the present study, combination of gliclazide and sildenafil, gliclazide and vinpocetine, vinpocetine and sildenafil increased insulin secretion in a concentration dependent manner in presence of basal or stimulatory glucose levels. Also, combination of gliclazide and sildenafil, gliclazide and vinpocetine, vinpocetine and sildenafil increased cGMP level in a concentration dependent manner in presence of basal or stimulatory glucose.

Our study demonstrated that there is a positive correlation between insulin release and cGMP level in isolated pancreatic islets in presence of individual or combined drugs. The study confirmed the hypothesis that, cGMP and cAMP may have a role in the augmentation of insulin secretion (Ishikawa et al., 2003; Smukler et al., 2002). The glucose stimulation threshold requires minimum cAMP concentration generated by the

11 same rate of glucose metabolism indicating the dependence of insulin secretion on a minimum concentration of cAMP (Weinhaus et al., 1998).

The insulinotropic effect of cGMP can be attributed to its effect on cytosolic Ca2+ concentration of rat pancreatic islets (Ishikawa et al., 2003). cGMP may increase insulin

2+ secretion owing to its inhibition of KATP channel and facilitation of Ca influx through L- type voltage operated Ca2+ channel (Ishikawa et al., 2003; Kaneko et al., 2003).

There is a relation between cGMP and cAMP actions where cGMP can generate cAMP or can mimic the cAMP’s stimulatory actions on insulin secretion. cGMP stimulates insulin release by a cross-specific activation of cAMP-protein kinase A where cGMP binds to the same sites of cAMP (Forte et al., 1992; Schumacher et al.,

1992). Dual regulation of PKA and Epac-Rap1 by cAMP and cGMP has been described by Cornwell et al (Cornwell et al., 1994).

PDE 1A and PDE 1C appear to hydrolyze both cAMP and cGMP in intact pancreatic cells (Miller et al., 2011; Pyne et al., 2003). Inhibition of PDE1 in pancreatic islets elevates the level of both cyclic nucleotides (cAMP and cGMP) which sequentially augments insulin release in presence of glucose.

There is another assumption that PDE1 and PDE5 are not predominant in pancreatic islets and their inhibition is not effective in augmenting insulin secretion. The predominant isozyme is PDE3 (Bender et al., 2006; Heimann et al., 2010). However,

PDE 3 hydrolysis of cAMP is inhibited by cGMP. The increased cGMP can be achieved through inhibition of PDE 1 and PDE 5 in pancreatic islets. Inhibition of cAMP hydrolysis increases the level of cAMP and evokes both PKA dependent and independent pathways for pancreatic insulin secretion (Bender et al., 2006).

12 cAMP potentiate glucose induced insulin secretion by 2 mechanisms; protein kinase A dependent and protein kinase A independent mechanisms (Seino, 2012).

Light et al (2002) stated that PKA-dependent mechanism includes phosphorylation of sulphonylurea receptor (Sur 1) of potassium channel. This phosphorylation decreases K+ channel’s activity and intiates the membrane depolarization.

The PKA-independent mechanism is mediated by exchange protein directly activated by cAMP (Epac) family. Epac is a guanine nucleotide exchange factor (GEF) for the low molecular weight GTP binding protein Rap1 (Kawasaki et al., 1998). Rap has been shown to control a number of cellular processes. Rap1 stimulates the recruitment of secretory granules to the membrane and activation of phospholipase-C in pancreatic beta- islets(Dzhura et al., 2010; Tian et al., 2012).

The 2 variants Epac namely; Epac 1 and Epac 2 where they mediate the stimulatory actions of secondary messenger cAMP on insulin secretion from pancreatic β-cells

2+ (Kang et al., 2006). Epac 2 inhibits KATP channels, activates ryanodine-sensitive Ca channel (Ryr 2), that is involved in Ca2+-induced-Ca2+-release (CICR) from endoplasmic reticulum, and participates in the spatial regulation of insulin granules exocytosis

(Grapengiesser et al., 1991; Shibasaki et al., 2007).

These 2 mechanisms of cAMP ensure that; raising cAMP level increases electrical activity and Ca2+ level in intact islets or isolated β-cells stimulated by glucose

(Grapengiesser et al., 1991; Yada et al., 1993).

Cyclic nucleotides (CN) stimulates rate of tubulin synthesis which may accounts for augmented insulin release in fasted islet cells (Pipeleers et al., 1976).

In the present work, the combination of test drugs showed antagonism on insulin secretion in presence “data not shown”. The antagonism of insulin secretion can be rationalized by parallel antagonism of cGMP level after addition of combined test drugs.

The drugs generate cGMP formation to certain level by inhibiting phosphodiesterase enzyme. After this level, negative feedback mechanisms lower the elevated cGMP level.

Increased cGMP level increases its degradation according to mass action effect and through phosphorylating PDE which increase cGMP binding to allosteric site stimulating cGMP degradation by the catalytic site of PDE (Corbin, 2004).

Ishikawa et al (2003) showed that cGMP causes rapid Ca2+ sequestration in endoplasmic reticulum through activating Ca2+-ATPase on the endoplasmic reticulum (Karaki et al.,

1997). Through this action, cGMP protects the pancreatic islets from excessive Ca2+- induced apoptosis (Efanova et al., 1998).

Finally, according to the findings of the present study, it could be concluded that PDEIs have been augmented insulin release due to elevation of cyclic nucleotides level. The level of cyclic nucleotides is directly proportional to insulin release. The PDEIs showed antagonism to gliclazide’s insulinotropic effect owing to their contributionsto elevate cGMP which concurrently stimulate negative feedback mechanisms that counteract the elevated cGMP level.

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Figures and Tables Tab. (1): Effects of different concentrations of gliclazide, vinpocetine or sildenafil alone on insulin secretion in presence of glucose (3 mmol/l) from isolated pancreatic islets of rats. In presence of glucose (3 mmol/l) Treatments Insulin secretion/islet/1 hr % of control ± S.E. (µIU/ml) Basal glucose (3 mmol/l) 1.867± 0.127 100 10 µmol/l * 3.775± 0.088 202.19 20 µmol/l * 4.475± 0.038 239.68 Gliclazide 40 µmol/l * 4.967± 0.112 266.04 80 µmol/l * 6.008 ± 0.058 321.8 10 µmol/l a1.725± 0.105 92.39 a Vinpocetine 20 µmol/l 1.975± 0.083 105.78 40 µmol/l *a2.750± 0.246 147.29 80 µmol/l *ab 4.442 ± 0.241 237.92 a 10 µmol/l 1.317± 0.073 70.54 a Sildenafil 20 µmol/l 1.908 ± 0.082 102.2 40 µmol/l *a 2.383 ± 0.080 127.63 80 µmol/l *a 2.758 ± 0.168 147.72 Number of experiments in each group = 6 Data were expressed as mean ± S.E. Statistical analysis was carried out by 2 ways repeated measures ANOVA followed by Post-test Newmans-Keuls multiple comparison test *: Significantly different from normal control at P<0.05 a: Significantly different from the corresponding gliclazide concentration at P<0.05 b: Significantly different from the corresponding sildenafil concentration at P<0.05.

Glic. + Silden. (10, 20, 40, 80 µmol/l) Glic. + Vinp. (10, 20, 40, 80 µmol/l)

Vinp. + Silden.(10, 20, 40, 80 µmol/l) Basal glucose (3 mmol/l) * 6 *a * * *ab * * * 4 * *ab *ab

/ (µIU ml) ab 2

/ /hr islet 1 secretion Insulin 0 10 20 30 40 50 60 70 80 90 Concentrations of drugs

(µMol/l)

Fig. (2): Effects of different concentrations of combined treatments of gliclazide and vinpocetine or sildenafil as well as vinpocetine and sildenafil on insulin secretion in presence of glucose (3 mmol/l) from isolated pancreatic islets of rats. Number of experiments in each group = 6 Data were expressed as mean ± S.E. Statistical analysis was carried out by 2 ways repeated measures ANOVA followed by Post-test Newmans-Keuls multiple comparison test *: Significantly different from normal control at P<0.05. a: Significantly different from the corresponding gliclazide and sildenafil concentration at P<0.05. b: Significantly different from the corresponding gliclazide and vinpocetine concentration at P<0.05.

Gliclazide (10, 20, 40, 80 µmol/l) Vinpocetine (10, 20, 40, 80 µmol/l) Sildenafil (10, 20, 40, 80 µmol/l) Stimulatory glucose (16.7 mmol/l) * 30 *

* * *ab 20 *ab *ab *ab *a

(µIU/ml) *a *a 10 *a

0 Insulin secretion / /islet hr 1 secretion Insulin 0 10 20 30 40 50 60 70 80 90 Concentrations of drugs (µMol/l)

Fig. (3): Effects of different concentrations of gliclazide, vinpocetine, sildenafil alone on insulin secretion in presence of glucose (16.7 mmol/l) from isolated pancreatic islets of rats. Number of experiments in each group = 6 Data were expressed as mean ± S.E. Statistical analysis was carried out by 2 ways repeated measures ANOVA followed by Post-test Newmans-Keuls multiple comparison test *: Significantly different from normal control at P<0.05. a: Significantly different from the corresponding gliclazide concentration at P<0.05. b: Significantly different from the corresponding sildenafil concentration at P<0.05.

Tab. (4): Effects of different concentrations of combined treatments of gliclazide and vinpocetine or sildenafil as well as vinpocetine and sildenafil on insulin secretion in presence of glucose (16.7 mmol/l) from isolated pancreatic islets of rats. In presence of glucose (16.7 mmol/l) Treatments Insulin secretion/islet/1 hr % of control ± S.E. (µIU/ml) Stimulatory glucose (16.7 mmol/l) 7.383 ± 0.671 100 * 10 µmol/l + 10 µmol/l 20.292 ± 0.643 274.84 Gliclazide * + 20 µmol/l + 20 µmol/l 23.85 ± 0.578 323.03 * Sildenafil 40 µmol/l + 40 µmol/l 25.433 ± 0.613 344.48 * 80 µmol/l + 80 µmol/l 27.133 ± 0.512 367.5 * 10 µmol/l + 10 µmol/l 22.175 ± 0.488 300.35 Gliclazide * + 20 µmol/l + 20 µmol/l 23.833 ± 0.832 322.8 * Vinpocetine 40 µmol/l + 40 µmol/l 25.25 ± 0.85 342 * 80 µmol/l + 80 µmol/l 27.941 ± 0.829 378.4 *b 10 µmol/l + 10 µmol/l 18.7 ± 0.752 253.28 Sildenafil *ab + 20 µmol/l + 20 µmol/l 20.683 ± 0.823 280.14 *ab Vinpocetine 40 µmol/l + 40 µmol/l 21.742 ± 1.09 294.48 *ab 80 µmol/l + 80 µmol/l 22.967 ± 1.016 311.08 Number of experiments in each group = 6 Data were expressed as mean ± S.E. Statistical analysis was carried out by 2 ways repeated measures ANOVA followed by Post-test Newmans-Keuls multiple comparison test *: Significantly different from normal control at P<0.05. a: Significantly different from the corresponding gliclazide and sildenafil concentration at P<0.05. b: Significantly different from the corresponding gliclazide and vinpocetine concentration at P<0.05.

Tab. (5): Effects of different concentrations of gliclazide, vinpocetine or sildenafil alone on cGMP level in presence of glucose (3 mmol/l) from isolated pancreatic islets of rats. In presence of glucose (3 mmol/l) Treatments cGMP level/islet/1 hr ± S.E. % of control (nmol/l) Basal glucose (3 mmol/l) 0.465± 0.023 100 * Gliclazide 10 µmol/l 0.820 ±0.028 176.34 * 20 µmol/l 1.147 ±0.033 246.67 * 40 µmol/l 1.300 ±0.033 279.56 * 80 µmol/l 1.685 ±0.095 362.36 a Vinpocetine 10 µmol/l 0.638 ±0.045 137.2 *a 20 µmol/l 0.717 ±0.04 154.2 *a 40 µmol/l 0.797 ±0.058 171.4 *ab 80 µmol/l 1.065 ±0.037 229.03 a Sildenafil 10 µmol/l 0.483 ±0.043 103.87 a 20 µmol/l 0.612 ±0.033 131.62 *a 40 µmol/l 0.705 ±0.099 151.61 *a 80 µmol/l 0.875 ±0.062 188.17 Number of experiments in each group = 6 Data were expressed as mean ± S.E. Statistical analysis was carried out by 2 ways repeated measures ANOVA followed by Post-test Newmans-Keuls multiple comparison test *: Significantly different from normal control at P<0.05. a: Significantly different from the corresponding gliclazide concentration at P<0.05. b: Significantly different from the corresponding sildenafil concentration at P<0.05.

Glic. + Silden. (10, 20, 40, 80 µmol/l)

Glic. + Vinp. (10, 20, 40, 80 µmol/l)

Vinp. + Silden.(10, 20, 40, 80 µmol/l) Basal glucose (3 mmol/l) 2.0 *

*a 1.5 * * * *a 1.0 *ab *ab * (nmol / l) (nmol *b *ab 0.5

//hr islet 1 level cGMP 0.0 0 10 20 30 40 50 60 70 80 90 Concentrations of drugs (µMol / l)

Fig. (6): Effects of different concentrations of combined treatments of gliclazide and vinpocetine or sildenafil as well as vinpocetine and sildenafil on cGMP level in presence of glucose (3 mmol/l) from isolated pancreatic islets of rats. Number of experiments in each group = 6 Data were expressed as mean ± S.E. Statistical analysis was carried out by 2 ways repeated measures ANOVA followed by Post-test Newmans-Keuls multiple comparison test *: Significantly different from normal control at P<0.05. a: Significantly different from the corresponding gliclazide and sildenafil concentration at P<0.05. b: Significantly different from the corresponding gliclazide and vinpocetineconcentration at P<0.05.

Gliclazide (10, 20, 40, 80 µmol/l) Vinpocetine (10, 20, 40, 80 µmol/l) Sildenafil (10, 20, 40, 80 µmol/l) Stimualtory glucose (16.7 mmol/l) * 30 * *b

*b * 20 * *b *a *a a

(nmol / l) (nmol a 10

/ / hr islet 1 level cGMP 0

0 10 20 30 40 50 60 70 80 90

Concentrations of drugs (µMol / l)

Fig. (7): Effects of different concentrations of gliclazide, vinpocetine or sildenafil alone on cGMP level in presence of glucose (16.7 mmol/l) from isolated pancreatic islets of rats. Number of experiments in each group = 6 Data were expressed as mean ± S.E. Statistical analysis was carried out by 2 ways repeated measures ANOVA followed by Post-test Newmans-Keuls multiple comparison test *: Significantly different from normal control at P<0.05. a: Significantly different from the corresponding gliclazide concentration at P<0.05. b: Significantly different from the corresponding sildenafil concentration at P<0.05.

Glic. + Silden. (10, 20, 40, 80 µmol/l)

Glic. + Vinp. (10, 20, 40, 80 µmol/l) Vinp. + Silden.(10, 20, 40, 80 µmol/l) Stimulatory glucose (16.7 mmol/l) 50 * * * *ab 40 *ab *ab * 30 *b* *a

*a *a *a

20 (nmol / l) (nmol

cGMP level / / hr islet 1 level cGMP 0 0 10 20 30 40 50 60 70 80 90 Concentrations of drugs (µMol / l)

Fig. (8): Effects of different concentrations of combined treatments of gliclazide and vinpocetine or sildenafil as well as vinpocetine and sildenafil on cGMP level in presence of glucose (16.7 mmol/l) from isolated pancreatic islets of rats. Number of experiments in each group = 6 Data were expressed as mean ± S.E. Statistical analysis was carried out by 2 ways repeated measures ANOVA followed by Post-test Newmans-Keuls multiple comparison test. *: Significantly different from normal control at P<0.05. a: Significantly different from the corresponding Gliclazide and Sildenafil concentration at P<0.05. b: Significantly different from the corresponding Gliclazide and Vinpocetine concentration at P<0.05.