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THE EFFECTS OF CHRONIC L -NAME AND L -ARGININE ADMINISTRATION ON β-ADRENERGIC RESPONSIVENESS OF STZ-DIABETIC RAT ATRIA
Pharmacological Research, Vol. 41, No. 5, 2000 doi:10.1006rphrs.1999.0623, available online at http:rrwww.idealibrary.com on
THE EFFECTS OF CHRONIC L -NAME AND L -ARGININE ADMINISTRATION ON b-ADRENERGIC RESPONSIVENESS OF STZ-DIABETIC RAT ATRIA U. DENIZ DINCERaU , A. TANJU OZCELIKAY a and EMINE DEMIREL YILMAZ b a
Department of Pharmacology, Faculty of Pharmacy, Uni¨ ersity of Ankara, Tandogan, 06100 Ankara, Turkey and bDepartment of Pharmacology, Faculty of Medicine, Uni¨ ersity of Ankara, Sihhiye, 06100 Ankara, Turkey Accepted 22 October 1999
Recent studies have shown that NO acts as a negative inotrope and chronotrop in cardiac muscle. In the present study, we investigated the effects of the chronic administration of L-NAME and L-arginine on 14-week streptozotocin ŽSTZ.-diabetic rat atria. To study these effects, we compared the alterations of inotropic and chronotropic responses to isoprenaline ŽISO. on electrically-driven left atria and spontaneously beating right atria. In addition, we compared the blood pressures of rats in all groups. The chronic administration of L-arginine resulted in a significant reduction in blood pressure of the diabetic rats. On the other hand, the chronic nitric oxide synthase inhibition resulted in a significant increase in blood pressure of diabetic animals. To our findings, maximum positive inotropic responses of ISO diminished in STZ-diabetic, L-arginine and L-NAME treated diabetic groups relative to controls but neither the basal contractility of the left atria nor the pD2 values were altered significantly in all groups. The basal atrial rate and maximum positive chronotropic responses to ISO were found to be significantly lower in treated and untreated diabetic groups, no significant changes were observed in pD2 values. Our results demonstrate that the changes in inotropic and chronotropic responses in diabetic rat atria were not influenced by the chronic administration of L-arginine and L-NAME treatments. Q 2000 Academic Press KEY
WORDS:
diabetes, L-arginine and L-NAME treatments, rat right and left atria.
INTRODUCTION Nitric oxide ŽNO., an unstable radical, is synthesized from L-arginine by the constitutive ŽcNOS. and inducible ŽiNOS. forms of nitric oxide synthase ŽNOS. and acts as a negative inotrope in cardiac muscle w1x. In the sinus node, the stimulation of the M 2 receptor leads to formation of cGMP and the lessening adrenergic effect w2x. At the same time, cholinergic stimulation induces the activity of the myocardial cNOS which leads to the increased formation of NO w3x. In the myocardium, cGMP stimulates protein kinase G w4x, and, therefore, decreases the heart rate and induces the negative inotropic effect w5x. NO can be synthesized in the endocardial endothelial cells w6x and myocardial cells w7x by cNOS. Nevertheless, if cardiac myocytes w7x andror endocardium w8x are U
Corresponding author.
1043]6618r00r050565]06r$35.00r0
exposed to inflammatory cytokines or endotoxaemia, iNOS is synthesized which eventually leads to decreased contractile function. iNOS can be the dominant enzyme when activated during immune-related disorders such as myocarditis, allograft rejection and myocardial infarction or different types of heart failure. While this is so, excess NO can be produced in the myocardium or endocardium by iNOS when the pathologic situations developed. Habib et al. w9x reported that excess NO is produced in the myocardium in dilated cardiomyopathy when iNOS is stimulated. However, the role of NO on inotropic andror choronotropic response in different types of heart failure has not been investigated completely. The diabetic cardiomyopathy was recognized by Rubler et al. w10x in 1972 and it was independent of coronary artery complications. Attempts have been made to explain diastolic dysfunction which sometimes can be attributed to an altered matrix, as well as systolic mechanical abnormalities, Q 2000 Academic Press
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electrophysiological changes and the other alterations in clinical and experimental settings in the diabetic heart w11x. Several studies revealed a significant enhancement of interstitial collagen in the myo-cardium and a decreased diastolic compliance without ventricular hypertrophy or evidence of ischemia w12, 13x. On the other hand, beta-adrenergic stimulation characteristics of cardiac function of diabetic rats have been demonstrated to be depressed. Studies with experimentally-induced diabetic animals revealed bradycardia, decreased inotropic and chronotropic responses with b-adrenoceptor agonists w14]17x and a decreased number of cardiac b-adrenoceptors w18, 19x. The overall goal of this study was, therefore, to investigate whether or not the long-term NOS inhibition with L-NAME has any beneficial effect on defective beta-adrenergic responsiveness of streptozotocin-diabetic rat atria.
MATERIALS AND METHODS
Experimental animals Experimental diabetes was produced in lightly anaesthetized male albino rats Ž170]220 g. with ether by a single intravenous injection of streptozotocin ŽSTZ, 50 mg kgy1 . into the lateral tail vein. STZ was dissolved in 0.1 M citrate buffer ŽpH 4.5.. Control rats were injected with only the same citrate buffer and served as age-matched control group. One week later, blood glucose levels were determined using an Ames glucometer ŽGlucometer III, Bayer Diagnostics, France. and blood glucose test strips ŽGlucofilm, Bayer Diagnostics, Germany.. Rats with blood glucose levels of 300 mg dly1 or above were considered to be diabetic. All groups of animals were housed in individual cages with controlled temperature Ž228C. and care being taken with a fixed 12:12-h light]dark cycle Žlights on 07:00]19:00 h.. L -arginine
and L -NAME treatment
Seven days after the STZ injection, the diabetic rats were divided into three subgroups: one group was given plain tap water Žuntreated diabetics.; the other group was given drinking water containing y1 L-arginine Ž1 mg ml ; L-arginine treated diabetic group.; and the last group was given drinking water containing L-NAME Ž20 mg kgy1 ; L-NAME treated diabetic group.. The rats were maintained for 14 weeks with free access to food and water. All rats were killed 14 weeks after diabetes induction.
Measurement of systolic blood pressure Systolic blood pressure was measured in conscious rats by the tail-cuff method before STZ or citrate buffer injection, at 14 weeks. All rats were preconditioned to the experimental conditions before actual
measurements were conducted. At the time of experiment, the rats were placed in a constant temperature Ž328C. chamber for 90 min. Thereafter, the animals were put in a rat holder. The tail-cuff and pulse sensor were placed on the tail and were connected to a semiautomatic blood pressure analyser ŽMAY 9610, Commat Ltd., Ankara, Turkey.. Since the rats had been preconditioned to the blood pressure measurement procedure, they became calm during the measurements. Eight individual readings were obtained within 10 min. The highest and the lowest measurements were discarded, and the average of the remaining six readings was taken as the individual systolic blood pressure.
Plasma analysis Deep anaesthesia with intraperitoneally administered sodium tiopenthal ŽAbbott, 60 mg kg -1 . was induced, approximately 4]5 ml of blood was collected in heparinized tubes through a small incision in the left arteria renalis. Blood samples were immediately centrifuged Ž3000 g for 20 min. and plasma was collected and stored at y308C until assay. Final plasma glucose levels were determined by the glucose oxidation method using the Menarini diagnostic glucose kit ŽGlucofix.. Menari diagnostic kits were also used to measure the levels of cholesterol and triglyceride in the plasma. The cholesterol and triglyceride levels were determined using enzymatic and colorimetric methods on a programmable photometer Žfototron, diatron, Wien, Austria.. Plasma insulin was measured by standard radioimmunoassay techniques using a commercial kit available from DPC ŽDiagnostic Products Corporation, Los Angeles, CA, USA..
Tissue isolation After taking blood samples the chest of the rat was opened and the heart was quickly excised and the right and left atria were carefully dissected from the ventricles and remaining non-atrial tissues. The vena cava superior was connected to a tissue holder and the appendix of the right atria was connected to an isometric transducer ŽUgo Basile, No.7006. and recording on an Ugo Basile microdynamometer ŽNo. 7005.. Left atria was suspended vertically between bipolar platinum electrodes and electrically stimulus applied at a frequency of 1 Hz with rectangular wave pulses of 1-ms duration at a voltage approximately 20% above threshold ŽGrass Stimulator S44.. The preparation being set up in a 20-ml organ bath containing Krebs-bicarbonate solution used at 378C ŽpH 7.4..The composition of the Krebs-bicarbonate solution was ŽmM.: NaCl, 118.4; KCI, 4.7, CaCl 2 ? 6H 2 O, 2.5; KH 2 PO4 , 1.2; MgSO4 ?7H 2 O, 1.2; Na HCO 3 , 22.3 and glucose, 5.6 and oxygenated with 95% O 2 q5% CO 2 gas mixture. The atrial tissue was subjected to a resting tension of 1 g.
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Experimental procedure The tissues were allowed to equilibrate for 60 min. After ensuring steady base-line recordings, the tissues were sensitized by a brief exposure to Ž5 = 10y6 M. ISO. The tissues were again washed every 15 min in order to obtain steady baselines, and then cumulative concentration-effect curve of ISO was performed. Agonist responses were measured as the beat per minute of the right atrium and increase in the percentage of tension of the left atrium.
Drugs The drugs used in this study were as follows: Streptozotocin ŽSigma., isoproterenol ŽSigma., Larginine hydrochloride ŽSigma. and N v-Nitro-LArginine Methyl Ester ŽL-NAME. ŽSigma.. All the drug stock solutions were freshly prepared.
Data analysis and statistics
The data are shown as mean " SE of the mean ŽSEM.. Results were considered significantly different when P- 0.05. Statistical analysis was performed according to one-way analysis of variance ŽANOVA. followed by Neuman]Keul’s test. Agonist pD2 values Žapparent agonist affinity constant; ylog ED50 . shown in Table II and Table III were calculated using non-linear regression analysis on the curve in Figs 1 and 2 and taken as a measure of the sensitivity of the tissues to ISO.
RESULTS
Body weight and plasma concentrations of glucose, insulin, cholesterol and triglycerides After STZ treatment, rats demonstrated some of the characteristics of insulin-dependent diabetes, namely polyphagia, polyuria, hypoinsulinemia and stable hyperglycemia. Fourteen weeks following STZ injections, body weights of untreated diabetic, Larginine and L-NAME treated diabetic groups were significantly less than that of the control group Ž P0.05. ŽTable I.. On the other hand, diabetic rats had significantly higher levels of plasma glucose, triglycerides and cholesterol, and significantly lower plasma insulin concentration than those of control rats Ž P0.05. ŽTable I.. L-arginine and L-NAME treatments did not alter plasma glucose, insulin, cholesterol and triglyceride levels ŽTable I..
Fig. 1. The positive chronotropic effects of isoprenaline of spontaneously beating right atria from control ŽB., diabetic Žv., L-arginine treated diabetic Žl. and L-NAME treated diabetic Ž'. rats. Values are means " SEM. U P0.05 significantly different from all other groups.
nificant increase of blood pressure in the diabetic group when compared with other groups Ž P- 0.05. ŽTable I..
Positi¨ e chronotropic responses to isoprenaline of isolated right atria ISO caused a dose-dependent increase in heart rate in all groups ŽFig. 1.. Basal atrial rate and maximum positive chronotropic responses to isoprenaline ŽISO. were found to be significantly lower in diabetic, L-arginine and L-NAME-treated diabetic groups than those of control animals ŽTable II.. However, neither the basal atrial rate nor maximum rate of atrial response to ISO were altered with L-arginine and L-NAME treatments of STZ-diabetic rats. L-arginine and L-NAME treatment did not alter the chronotropic effect of ISO in diabetic animals.
Changes in systolic blood pressures Fourteen weeks after STZ administration, the systolic blood pressure was increased in diabetic and L-NAME treated diabetic groups when compared with control and L-arginine treated diabetic group Ž P- 0.05.. The chronic administration of L-arginine resulted in a significant reduction of blood pressure in the diabetic animals Ž P- 0.05.. On the other hand, the chronic NOS inhibition resulted in a sig-
Fig. 2. The positive inotropic effects of isoprenaline of electricaly stimulated left atria from control ŽB., diabetic Žv., L-arginine treated diabetic Žl. and L-NAME treated diabetic Ž'. rats. Values are means " SEM. U P - 0.05 significantly different from all other groups.
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568
Table I Some characteristics of control, untreated, L-arginine treated and L-NAME treated diabetic rats
Body weight Žg. Systolic blood pressures ŽmmHg. Plasma glucose Žmg dly1 . Plasma insulin Žpmol ly1 . Plasma cholesterol Žmg dly1 . Plasma triglyceride Žmg dly1 .
Control (n s 5)
Untreated Diabetic (n s 6)
L-arg
treated diabetic (n s 7)
L-NAME
treated Diabetic (n s 7)
359.6" 18.6 117.9" 2.7
213.6" 10.3U 150.1" 4.9U ,UU
218.2" 13.2U 124.5" 3.8
180.8" 14.8U 164.7" 4.9U ,UU ,UUU
115.7" 3.9
391.9" 7.3U
397.2" 4.6U
380.2" 8.5U
191.2" 30.3
39.7" 4.1U
39.2" 5.0U
35.6" 6.6U
56.1" 3.3
71.0" 4.4U
75.5" 5.5U
78.6" 13.3U
91.8" 3.1
172.8" 19.64U
170.4" 24.6U
159.9" 32.7U
Note. Values are expressed as mean " SEM. U P- 0.05 significantly different from control group. UU P - 0.05 significantly different from L-arginine treated diabetic group. UUU P- 0.05 significantly different from untreated diabetic group.
pD2 value calculated for the positive chronotropic effect of ISO in all groups,however, were found to be unchanged.
inotropic effect of ISO were not altered significantly in all groups ŽTable III..
Positi¨ e inotropic responses to ISO of isolated left atria
DISCUSSION
ISO produced concentration-dependent positive inotropic effects in all groups ŽFig. 2.. Maximum positive inotropic responses to ISO were found significantly lower in untreated diabetic, L-arginine and L-NAME-treated diabetic groups when compared with control rats, but neither the basal contractility of left atria nor the pD2 values for the positive
Diabetes, as expected, caused hyperglycaemia, hypoinsulinemia, enhanced plasma cholesterol and triglyceride levels, decreased body weight and depressed cardiac function in rats. All these changes were found to be similar in L-arginine and L-NAME treated diabetic groups. The chronic administration of L-arginine resulted in a significant reduction in
Table II Basal heart rate, maximum chronotropic responses and pD 2 values for isoprenaline on right atria
Control Untreated diabetic L-arginine treated diabetic L-NAME treated diabetic
n
Basal rate (b.p.m.)
Maximum response (b.p.m.)
pD2
6 5 6 6
191.7" 27.0 124.8" 20.4U 91.7" 5.4U 101.7" 10.1U
361.7" 30.3 278.8" 19.8U 288.3" 8.7U 271.7" 17.8U
6.99" 0.14 6.72" 0.19 6.76" 0.12 6.21" 0.33
Note. Values are expressed as mean " SEM. U P - 0.05 significantly different from control group.
Table III Basal tension, % increase of maximum inotropic responses and pD 2 values for isoprenaline on left atria
Control Untreated diabetic L-arginine treated diabetic L-NAME treated diabetic
n
Basal tension (mg)
Maximum response (% increase)
pD2
5 6 6
256.8" 41.4 328.8" 55.6 256.8" 52.1
111.2" 10.0 49.4" 7.5a 80.0" 12.0a
7.11" 0.43 7.10" 0.22 6.81" 0.31
5
227.0" 35.9
70.7" 6.9a
6.50" 0.26
Note. Values are expressed as mean " SEM. U P- 0.05 significantly different from the control group.
Pharmacological Research, Vol. 41, No. 5, 2000
blood pressure of diabetic rats while chronic NOS inhibition resulted in a significant increase in blood pressure of those rats. Maree et al. w20x also found the same results in 6 weeks in STZ-diabetic rats. In keeping with our results, many studies have revealed that moderate or severe NOS inhibition Žgiven LNAME in drinking water. leads to an increase in blood pressure w21]23x. In the present study, chronic L-NAME treatment did not influence the decreased inotropic and chronotropic responses obtained with ISO in the diabetic rat hearts. Various studies have demonstrated that L-NAME treatment causes increased blood pressure but that the results of cardiac hypertrophic effects are not always consistent. Devlin et al. w24x showed that in ¨ i¨ o NOS inhibition caused a significant increase in cardiac hypertrophy together with hypertension using L-NAME. On the other hand, Moreno et al. w25x found that chronic inhibition of NO biosynthesis causes cardiac ischemia associated with a mechanical dysfunction that is unrelated to cardiac hypertrophy. In the same way, Wickman et al. w23x revealed that L-NAME treatment caused hypertension which led neither to left ventricular hypertrophy nor to the expected overexpression of the left ventricularraortic insulin-like growth factor-1 mRNA. The decreased inotropic and chronotropic responsiveness of diabetic hearts have been extensively investigated. Many investigators have revealed deficiencies in beta adrenergic responses and receptors w16, 18, 19x in the diabetic heart. In a recent study, we found that the maximum chronotropic responses of the right atria from 14-week diabetic rats decreased 25 and 29% when stimulated with ISO and noradrenaline, respectively w17x. After the b-adrenergic stimulation, the b-adrenoceptors coupled to adenyl cyclase by the stimulatory G protein, Gs . The adenyl cyclase is activated by Gs and inhibited by Gi . However, in the disease states, their role is not completely clear. Gando et al. also demonstrated that the expression of Gi was markedly diminished but Gs remained unchanged in the diabetic myocardium w26x. On the other hand, Yamamato et al. reported that the increased NO induction attenuates the positive inotropic response to ISO in failing myocytes obtained from dogs w27x. They also suggested that NO plays an important role in the autocrine regulation of the contractile function of myocytes in congestive heart failure. Similarly, Smith et al. w28x found the presence of iNOS and a significant increase in the cNOS in ventricular myocytes from 8 weeks STZdiabetic rats using Western blot analysis. They also showed that increased production of NO by NOS in STZ-diabetic ventricular myocytes suppressed basal ventricular performance and the responsiveness to beta-adrenergic stimulation in diabetic hearts. In the same study, L-NAME infusion restored depressed heart rate and ventricular performance of diabetic
569
rats w28x. In our study, therefore, we hypothesised that treatment of L-NAME in ¨ i¨ o, a non-selective inhibitor of NOS, can alleviate but L-arginine administration, substrate for NO synthesis, can exacerbate the abnormalities in atria isolated from diabetic rats. However, our results showed that neither L-NAME treatment in contrast to findings of Smith et al. w28x nor L-arginine treatment has any effect on impaired atrial performance in diabetic rats. On the other hand, L-NAME treatment aggravated the hypertension in diabetic rats while L-arginine treatment kept systolic blood pressure down. Our results showed that although effects of L-NAME or L-arginine treatment occurred in the vascular system, they failed to affect in the atria of diabetic rats, suggesting that L-arginine NO pathway was not affected by Larginine and L-NAME at the doses used in this study. Thereby L-arginine and L-NAME was not responsible for decreased atrial performance in diabetic rats. However, Feng et al. w29x found that chronic oral L-arginine treatment Žat the dose of 12.5 times higher that we used. improved endothelium-dependent relaxation, but failed to improve in ¨ i¨ o cardiac function in rats with heart failure. Similarly, previous studies have shown that there was no effect of L-NAME on the left and right atrial preparations isolated from hearts of guinea pigs and rats, respectively w30, 31x. In conclusion, our results demonstrate that the changes in inotropic and chronotropic responses in diabetic rat atria were not influenced by the chronic administration of L-arginine and L-NAME treatments. Thus, NOS activation seems to have no contribution to the decreased responsiveness of diabetic atria to beta-adrenergic stimulation.
ACKNOWLEDGEMENTS This work was supported by the Eczacibasi Drug Company in Turkey.
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THE EFFECTS OF CHRONIC L -NAME AND L -ARGININE ADMINISTRATION ON b-ADRENERGIC RESPONSIVENESS OF STZ-DIABETIC RAT ATRIA U. DENIZ DINCERaU , A. TANJU OZCELIKAY a and EMINE DEMIREL YILMAZ b a
Department of Pharmacology, Faculty of Pharmacy, Uni¨ ersity of Ankara, Tandogan, 06100 Ankara, Turkey and bDepartment of Pharmacology, Faculty of Medicine, Uni¨ ersity of Ankara, Sihhiye, 06100 Ankara, Turkey Accepted 22 October 1999
Recent studies have shown that NO acts as a negative inotrope and chronotrop in cardiac muscle. In the present study, we investigated the effects of the chronic administration of L-NAME and L-arginine on 14-week streptozotocin ŽSTZ.-diabetic rat atria. To study these effects, we compared the alterations of inotropic and chronotropic responses to isoprenaline ŽISO. on electrically-driven left atria and spontaneously beating right atria. In addition, we compared the blood pressures of rats in all groups. The chronic administration of L-arginine resulted in a significant reduction in blood pressure of the diabetic rats. On the other hand, the chronic nitric oxide synthase inhibition resulted in a significant increase in blood pressure of diabetic animals. To our findings, maximum positive inotropic responses of ISO diminished in STZ-diabetic, L-arginine and L-NAME treated diabetic groups relative to controls but neither the basal contractility of the left atria nor the pD2 values were altered significantly in all groups. The basal atrial rate and maximum positive chronotropic responses to ISO were found to be significantly lower in treated and untreated diabetic groups, no significant changes were observed in pD2 values. Our results demonstrate that the changes in inotropic and chronotropic responses in diabetic rat atria were not influenced by the chronic administration of L-arginine and L-NAME treatments. Q 2000 Academic Press KEY
WORDS:
diabetes, L-arginine and L-NAME treatments, rat right and left atria.
INTRODUCTION Nitric oxide ŽNO., an unstable radical, is synthesized from L-arginine by the constitutive ŽcNOS. and inducible ŽiNOS. forms of nitric oxide synthase ŽNOS. and acts as a negative inotrope in cardiac muscle w1x. In the sinus node, the stimulation of the M 2 receptor leads to formation of cGMP and the lessening adrenergic effect w2x. At the same time, cholinergic stimulation induces the activity of the myocardial cNOS which leads to the increased formation of NO w3x. In the myocardium, cGMP stimulates protein kinase G w4x, and, therefore, decreases the heart rate and induces the negative inotropic effect w5x. NO can be synthesized in the endocardial endothelial cells w6x and myocardial cells w7x by cNOS. Nevertheless, if cardiac myocytes w7x andror endocardium w8x are U
Corresponding author.
1043]6618r00r050565]06r$35.00r0
exposed to inflammatory cytokines or endotoxaemia, iNOS is synthesized which eventually leads to decreased contractile function. iNOS can be the dominant enzyme when activated during immune-related disorders such as myocarditis, allograft rejection and myocardial infarction or different types of heart failure. While this is so, excess NO can be produced in the myocardium or endocardium by iNOS when the pathologic situations developed. Habib et al. w9x reported that excess NO is produced in the myocardium in dilated cardiomyopathy when iNOS is stimulated. However, the role of NO on inotropic andror choronotropic response in different types of heart failure has not been investigated completely. The diabetic cardiomyopathy was recognized by Rubler et al. w10x in 1972 and it was independent of coronary artery complications. Attempts have been made to explain diastolic dysfunction which sometimes can be attributed to an altered matrix, as well as systolic mechanical abnormalities, Q 2000 Academic Press
Pharmacological Research, Vol. 41, No. 5, 2000
566
electrophysiological changes and the other alterations in clinical and experimental settings in the diabetic heart w11x. Several studies revealed a significant enhancement of interstitial collagen in the myo-cardium and a decreased diastolic compliance without ventricular hypertrophy or evidence of ischemia w12, 13x. On the other hand, beta-adrenergic stimulation characteristics of cardiac function of diabetic rats have been demonstrated to be depressed. Studies with experimentally-induced diabetic animals revealed bradycardia, decreased inotropic and chronotropic responses with b-adrenoceptor agonists w14]17x and a decreased number of cardiac b-adrenoceptors w18, 19x. The overall goal of this study was, therefore, to investigate whether or not the long-term NOS inhibition with L-NAME has any beneficial effect on defective beta-adrenergic responsiveness of streptozotocin-diabetic rat atria.
MATERIALS AND METHODS
Experimental animals Experimental diabetes was produced in lightly anaesthetized male albino rats Ž170]220 g. with ether by a single intravenous injection of streptozotocin ŽSTZ, 50 mg kgy1 . into the lateral tail vein. STZ was dissolved in 0.1 M citrate buffer ŽpH 4.5.. Control rats were injected with only the same citrate buffer and served as age-matched control group. One week later, blood glucose levels were determined using an Ames glucometer ŽGlucometer III, Bayer Diagnostics, France. and blood glucose test strips ŽGlucofilm, Bayer Diagnostics, Germany.. Rats with blood glucose levels of 300 mg dly1 or above were considered to be diabetic. All groups of animals were housed in individual cages with controlled temperature Ž228C. and care being taken with a fixed 12:12-h light]dark cycle Žlights on 07:00]19:00 h.. L -arginine
and L -NAME treatment
Seven days after the STZ injection, the diabetic rats were divided into three subgroups: one group was given plain tap water Žuntreated diabetics.; the other group was given drinking water containing y1 L-arginine Ž1 mg ml ; L-arginine treated diabetic group.; and the last group was given drinking water containing L-NAME Ž20 mg kgy1 ; L-NAME treated diabetic group.. The rats were maintained for 14 weeks with free access to food and water. All rats were killed 14 weeks after diabetes induction.
Measurement of systolic blood pressure Systolic blood pressure was measured in conscious rats by the tail-cuff method before STZ or citrate buffer injection, at 14 weeks. All rats were preconditioned to the experimental conditions before actual
measurements were conducted. At the time of experiment, the rats were placed in a constant temperature Ž328C. chamber for 90 min. Thereafter, the animals were put in a rat holder. The tail-cuff and pulse sensor were placed on the tail and were connected to a semiautomatic blood pressure analyser ŽMAY 9610, Commat Ltd., Ankara, Turkey.. Since the rats had been preconditioned to the blood pressure measurement procedure, they became calm during the measurements. Eight individual readings were obtained within 10 min. The highest and the lowest measurements were discarded, and the average of the remaining six readings was taken as the individual systolic blood pressure.
Plasma analysis Deep anaesthesia with intraperitoneally administered sodium tiopenthal ŽAbbott, 60 mg kg -1 . was induced, approximately 4]5 ml of blood was collected in heparinized tubes through a small incision in the left arteria renalis. Blood samples were immediately centrifuged Ž3000 g for 20 min. and plasma was collected and stored at y308C until assay. Final plasma glucose levels were determined by the glucose oxidation method using the Menarini diagnostic glucose kit ŽGlucofix.. Menari diagnostic kits were also used to measure the levels of cholesterol and triglyceride in the plasma. The cholesterol and triglyceride levels were determined using enzymatic and colorimetric methods on a programmable photometer Žfototron, diatron, Wien, Austria.. Plasma insulin was measured by standard radioimmunoassay techniques using a commercial kit available from DPC ŽDiagnostic Products Corporation, Los Angeles, CA, USA..
Tissue isolation After taking blood samples the chest of the rat was opened and the heart was quickly excised and the right and left atria were carefully dissected from the ventricles and remaining non-atrial tissues. The vena cava superior was connected to a tissue holder and the appendix of the right atria was connected to an isometric transducer ŽUgo Basile, No.7006. and recording on an Ugo Basile microdynamometer ŽNo. 7005.. Left atria was suspended vertically between bipolar platinum electrodes and electrically stimulus applied at a frequency of 1 Hz with rectangular wave pulses of 1-ms duration at a voltage approximately 20% above threshold ŽGrass Stimulator S44.. The preparation being set up in a 20-ml organ bath containing Krebs-bicarbonate solution used at 378C ŽpH 7.4..The composition of the Krebs-bicarbonate solution was ŽmM.: NaCl, 118.4; KCI, 4.7, CaCl 2 ? 6H 2 O, 2.5; KH 2 PO4 , 1.2; MgSO4 ?7H 2 O, 1.2; Na HCO 3 , 22.3 and glucose, 5.6 and oxygenated with 95% O 2 q5% CO 2 gas mixture. The atrial tissue was subjected to a resting tension of 1 g.
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Experimental procedure The tissues were allowed to equilibrate for 60 min. After ensuring steady base-line recordings, the tissues were sensitized by a brief exposure to Ž5 = 10y6 M. ISO. The tissues were again washed every 15 min in order to obtain steady baselines, and then cumulative concentration-effect curve of ISO was performed. Agonist responses were measured as the beat per minute of the right atrium and increase in the percentage of tension of the left atrium.
Drugs The drugs used in this study were as follows: Streptozotocin ŽSigma., isoproterenol ŽSigma., Larginine hydrochloride ŽSigma. and N v-Nitro-LArginine Methyl Ester ŽL-NAME. ŽSigma.. All the drug stock solutions were freshly prepared.
Data analysis and statistics
The data are shown as mean " SE of the mean ŽSEM.. Results were considered significantly different when P- 0.05. Statistical analysis was performed according to one-way analysis of variance ŽANOVA. followed by Neuman]Keul’s test. Agonist pD2 values Žapparent agonist affinity constant; ylog ED50 . shown in Table II and Table III were calculated using non-linear regression analysis on the curve in Figs 1 and 2 and taken as a measure of the sensitivity of the tissues to ISO.
RESULTS
Body weight and plasma concentrations of glucose, insulin, cholesterol and triglycerides After STZ treatment, rats demonstrated some of the characteristics of insulin-dependent diabetes, namely polyphagia, polyuria, hypoinsulinemia and stable hyperglycemia. Fourteen weeks following STZ injections, body weights of untreated diabetic, Larginine and L-NAME treated diabetic groups were significantly less than that of the control group Ž P0.05. ŽTable I.. On the other hand, diabetic rats had significantly higher levels of plasma glucose, triglycerides and cholesterol, and significantly lower plasma insulin concentration than those of control rats Ž P0.05. ŽTable I.. L-arginine and L-NAME treatments did not alter plasma glucose, insulin, cholesterol and triglyceride levels ŽTable I..
Fig. 1. The positive chronotropic effects of isoprenaline of spontaneously beating right atria from control ŽB., diabetic Žv., L-arginine treated diabetic Žl. and L-NAME treated diabetic Ž'. rats. Values are means " SEM. U P0.05 significantly different from all other groups.
nificant increase of blood pressure in the diabetic group when compared with other groups Ž P- 0.05. ŽTable I..
Positi¨ e chronotropic responses to isoprenaline of isolated right atria ISO caused a dose-dependent increase in heart rate in all groups ŽFig. 1.. Basal atrial rate and maximum positive chronotropic responses to isoprenaline ŽISO. were found to be significantly lower in diabetic, L-arginine and L-NAME-treated diabetic groups than those of control animals ŽTable II.. However, neither the basal atrial rate nor maximum rate of atrial response to ISO were altered with L-arginine and L-NAME treatments of STZ-diabetic rats. L-arginine and L-NAME treatment did not alter the chronotropic effect of ISO in diabetic animals.
Changes in systolic blood pressures Fourteen weeks after STZ administration, the systolic blood pressure was increased in diabetic and L-NAME treated diabetic groups when compared with control and L-arginine treated diabetic group Ž P- 0.05.. The chronic administration of L-arginine resulted in a significant reduction of blood pressure in the diabetic animals Ž P- 0.05.. On the other hand, the chronic NOS inhibition resulted in a sig-
Fig. 2. The positive inotropic effects of isoprenaline of electricaly stimulated left atria from control ŽB., diabetic Žv., L-arginine treated diabetic Žl. and L-NAME treated diabetic Ž'. rats. Values are means " SEM. U P - 0.05 significantly different from all other groups.
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568
Table I Some characteristics of control, untreated, L-arginine treated and L-NAME treated diabetic rats
Body weight Žg. Systolic blood pressures ŽmmHg. Plasma glucose Žmg dly1 . Plasma insulin Žpmol ly1 . Plasma cholesterol Žmg dly1 . Plasma triglyceride Žmg dly1 .
Control (n s 5)
Untreated Diabetic (n s 6)
L-arg
treated diabetic (n s 7)
L-NAME
treated Diabetic (n s 7)
359.6" 18.6 117.9" 2.7
213.6" 10.3U 150.1" 4.9U ,UU
218.2" 13.2U 124.5" 3.8
180.8" 14.8U 164.7" 4.9U ,UU ,UUU
115.7" 3.9
391.9" 7.3U
397.2" 4.6U
380.2" 8.5U
191.2" 30.3
39.7" 4.1U
39.2" 5.0U
35.6" 6.6U
56.1" 3.3
71.0" 4.4U
75.5" 5.5U
78.6" 13.3U
91.8" 3.1
172.8" 19.64U
170.4" 24.6U
159.9" 32.7U
Note. Values are expressed as mean " SEM. U P- 0.05 significantly different from control group. UU P - 0.05 significantly different from L-arginine treated diabetic group. UUU P- 0.05 significantly different from untreated diabetic group.
pD2 value calculated for the positive chronotropic effect of ISO in all groups,however, were found to be unchanged.
inotropic effect of ISO were not altered significantly in all groups ŽTable III..
Positi¨ e inotropic responses to ISO of isolated left atria
DISCUSSION
ISO produced concentration-dependent positive inotropic effects in all groups ŽFig. 2.. Maximum positive inotropic responses to ISO were found significantly lower in untreated diabetic, L-arginine and L-NAME-treated diabetic groups when compared with control rats, but neither the basal contractility of left atria nor the pD2 values for the positive
Diabetes, as expected, caused hyperglycaemia, hypoinsulinemia, enhanced plasma cholesterol and triglyceride levels, decreased body weight and depressed cardiac function in rats. All these changes were found to be similar in L-arginine and L-NAME treated diabetic groups. The chronic administration of L-arginine resulted in a significant reduction in
Table II Basal heart rate, maximum chronotropic responses and pD 2 values for isoprenaline on right atria
Control Untreated diabetic L-arginine treated diabetic L-NAME treated diabetic
n
Basal rate (b.p.m.)
Maximum response (b.p.m.)
pD2
6 5 6 6
191.7" 27.0 124.8" 20.4U 91.7" 5.4U 101.7" 10.1U
361.7" 30.3 278.8" 19.8U 288.3" 8.7U 271.7" 17.8U
6.99" 0.14 6.72" 0.19 6.76" 0.12 6.21" 0.33
Note. Values are expressed as mean " SEM. U P - 0.05 significantly different from control group.
Table III Basal tension, % increase of maximum inotropic responses and pD 2 values for isoprenaline on left atria
Control Untreated diabetic L-arginine treated diabetic L-NAME treated diabetic
n
Basal tension (mg)
Maximum response (% increase)
pD2
5 6 6
256.8" 41.4 328.8" 55.6 256.8" 52.1
111.2" 10.0 49.4" 7.5a 80.0" 12.0a
7.11" 0.43 7.10" 0.22 6.81" 0.31
5
227.0" 35.9
70.7" 6.9a
6.50" 0.26
Note. Values are expressed as mean " SEM. U P- 0.05 significantly different from the control group.
Pharmacological Research, Vol. 41, No. 5, 2000
blood pressure of diabetic rats while chronic NOS inhibition resulted in a significant increase in blood pressure of those rats. Maree et al. w20x also found the same results in 6 weeks in STZ-diabetic rats. In keeping with our results, many studies have revealed that moderate or severe NOS inhibition Žgiven LNAME in drinking water. leads to an increase in blood pressure w21]23x. In the present study, chronic L-NAME treatment did not influence the decreased inotropic and chronotropic responses obtained with ISO in the diabetic rat hearts. Various studies have demonstrated that L-NAME treatment causes increased blood pressure but that the results of cardiac hypertrophic effects are not always consistent. Devlin et al. w24x showed that in ¨ i¨ o NOS inhibition caused a significant increase in cardiac hypertrophy together with hypertension using L-NAME. On the other hand, Moreno et al. w25x found that chronic inhibition of NO biosynthesis causes cardiac ischemia associated with a mechanical dysfunction that is unrelated to cardiac hypertrophy. In the same way, Wickman et al. w23x revealed that L-NAME treatment caused hypertension which led neither to left ventricular hypertrophy nor to the expected overexpression of the left ventricularraortic insulin-like growth factor-1 mRNA. The decreased inotropic and chronotropic responsiveness of diabetic hearts have been extensively investigated. Many investigators have revealed deficiencies in beta adrenergic responses and receptors w16, 18, 19x in the diabetic heart. In a recent study, we found that the maximum chronotropic responses of the right atria from 14-week diabetic rats decreased 25 and 29% when stimulated with ISO and noradrenaline, respectively w17x. After the b-adrenergic stimulation, the b-adrenoceptors coupled to adenyl cyclase by the stimulatory G protein, Gs . The adenyl cyclase is activated by Gs and inhibited by Gi . However, in the disease states, their role is not completely clear. Gando et al. also demonstrated that the expression of Gi was markedly diminished but Gs remained unchanged in the diabetic myocardium w26x. On the other hand, Yamamato et al. reported that the increased NO induction attenuates the positive inotropic response to ISO in failing myocytes obtained from dogs w27x. They also suggested that NO plays an important role in the autocrine regulation of the contractile function of myocytes in congestive heart failure. Similarly, Smith et al. w28x found the presence of iNOS and a significant increase in the cNOS in ventricular myocytes from 8 weeks STZdiabetic rats using Western blot analysis. They also showed that increased production of NO by NOS in STZ-diabetic ventricular myocytes suppressed basal ventricular performance and the responsiveness to beta-adrenergic stimulation in diabetic hearts. In the same study, L-NAME infusion restored depressed heart rate and ventricular performance of diabetic
569
rats w28x. In our study, therefore, we hypothesised that treatment of L-NAME in ¨ i¨ o, a non-selective inhibitor of NOS, can alleviate but L-arginine administration, substrate for NO synthesis, can exacerbate the abnormalities in atria isolated from diabetic rats. However, our results showed that neither L-NAME treatment in contrast to findings of Smith et al. w28x nor L-arginine treatment has any effect on impaired atrial performance in diabetic rats. On the other hand, L-NAME treatment aggravated the hypertension in diabetic rats while L-arginine treatment kept systolic blood pressure down. Our results showed that although effects of L-NAME or L-arginine treatment occurred in the vascular system, they failed to affect in the atria of diabetic rats, suggesting that L-arginine NO pathway was not affected by Larginine and L-NAME at the doses used in this study. Thereby L-arginine and L-NAME was not responsible for decreased atrial performance in diabetic rats. However, Feng et al. w29x found that chronic oral L-arginine treatment Žat the dose of 12.5 times higher that we used. improved endothelium-dependent relaxation, but failed to improve in ¨ i¨ o cardiac function in rats with heart failure. Similarly, previous studies have shown that there was no effect of L-NAME on the left and right atrial preparations isolated from hearts of guinea pigs and rats, respectively w30, 31x. In conclusion, our results demonstrate that the changes in inotropic and chronotropic responses in diabetic rat atria were not influenced by the chronic administration of L-arginine and L-NAME treatments. Thus, NOS activation seems to have no contribution to the decreased responsiveness of diabetic atria to beta-adrenergic stimulation.
ACKNOWLEDGEMENTS This work was supported by the Eczacibasi Drug Company in Turkey.
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