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Cardiovascular Research 1998 39(3):651-656; doi:10.1016/S0008-6363(98)00176-X
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Copyright © 1998, European Society of Cardiology

Impaired paracrine effect of endothelin-1 on vascular smooth muscle in streptozotocin-diabetic rats

Sheng-Qian Wu1 and Fai Tang*

Department of Physiology, Faculty of Medicine, University of Hong Kong, Hong Kong, China

* Corresponding author. Tel.: +852-2819-9269; Fax: +852-2855-9730; E-mail: ftang@hkucc.hku.hk

Received 27 November 1997; accepted 15 April 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: This study was to examine the effect of streptozotocin-induced diabetes on endothelin-1 and its receptors in the mesenteric artery and in the thoracic aorta. Methods: Diabetes was induced in SD rats by streptozotocin. Insulin was given subcutaneously. Endothelin-1 levels in the plasma, thoracic aorta and mesenteric artery were measured using radioimmunoassay. The Bmax and Kd values of endothelin-1 receptors in the mesenteric artery and in the thoracic aorta were analyzed using Scatchard plot analysis. Preproendothelin mRNA levels were examined using RT-PCR. Results: Endothelin-1 levels in the mesenteric artery (83.6±6.9 pg/mg protein) and in the thoracic aorta (73.9±8.2 pg/mg protein) increased in 2 week diabetic rats compared with both control (51.8±5.3, 46.3±5.9 pg/mg protein) and insulin treated rats (65.6±8.1, 48.1±4.2 pg/mg protein) but not in 4 week diabetic rats. There was no change in plasma endothelin-1 levels in these diabetic rats. The RT-PCR results indicated that preproendothelin mRNA levels in the mesenteric artery (0.38±0.02 vs 0.52±0.05 units) and in the thoracic aorta (0.45±0.06 vs 0.62±0.03 units) decreased in 4 week diabetic rats but not in 2 week diabetic rats. A significant increase in Kd and Bmax of endothelin receptors in the mesenteric artery and in the thoracic aorta was observed in both 2 week (about 70%) and 4 week (80–85%) diabetic rats. Insulin replacement reversed the effects of diabetes on endothelin-1 peptide contents, preproendothelin mRNA levels and the binding activity in the blood vessels. Conclusion: Increased endothelin peptide content with no change in mRNA or decreased mRNA levels with no change in peptide content together with increased receptor binding sites and affinities might imply a decrease in endothelin release and therefore an impaired paracrine effect of endothelin on vascular smooth muscles in these STZ-diabetic rats.

KEYWORDS Endothelin; Diabetes; Paracrine; Rat; Receptors; Vascular smooth muscle


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Endothelin-1 (ET-1), a 21-amino acid peptide produced primarily in endothelial cells [1]is the most powerful endogenous vasoconstrictor agent known to date [2]. Locally produced ET-1 exerts its effects in an autocrine and paracrine fashion to constrict blood vessels by acting on smooth muscle and to stimulate production of other agents such as endothelial-derived relaxing factors and contracting factors [2, 3]. There is evidence suggesting that the production of ET-1 is changed in diabetes both in human subjects and in animal diabetic models. Several authors demonstrated an elevation in circulating plasma ET-1 levels in diabetic patients [4–6], whereas others found no change in plasma ET-1 levels compared to those of healthy volunteers [7, 8]. One study demonstrated a reduced conversion of big ET-1 to ET-1 in patients with diabetes mellitus [9]. Both significant elevation [5]and lack of change [10]of plasma ET-1 levels were reported in STZ-diabetic rats.

There are many reports demonstrating functional changes in various smooth muscles from diabetic animals [11, 12]. These changes seem to be closely related to certain complications observed in the clinical diabetes, although the investigations on diabetic smooth muscles of diabetic animals have failed to produce consistent results. However, at least some of the changes may be related to the sensitivity of receptors to certain agonists and/or to altered number of receptors [13]. Unfortunately, due to methodological difficulties, direct assessment of receptors by radioligand binding assay has been made in only a few studies. Results obtained in the radioligand binding studies have revealed that experimental diabetes may cause both qualitative and quantitative changes in certain receptors in smooth muscles [13]. Reduced cardiovascular responsiveness to ET-1 was reported in blood vessels of diabetic animals with the exception of the increased responsiveness of the renal circulation. Attenuated responses to ET-1 were reported in aortae from rats with STZ-induced diabetes mellitus [14, 15]. Although the down-regulation of ET receptors have been reported in STZ-diabetic rat renal glomeruli [16]and heart muscle cell membranes [17], similar data are lacking for the rat vasculature.

Elevated, unchanged and even reduced plasma ET-1 levels have been reported in diabetic patients and streptozotocin-induced diabetic rats. However, because the release of ET-1 by endothelial cells is polarized toward the basolateral side [18], plasma levels of ET-1 may not correctly represent production rate, and the local concentration of the peptide might be much higher than in the plasma [19]. The role of circulating ET-1 in the regulation of vascular tone is possibly minor. Abluminal secretion of ET-1 plays a more important role through its autocrine and paracrine effects. Diabetes may affect the functioning of the vasculature through a change in ET production and/or receptor binding. The aims of this study are: [1]to investigate the ET-1 contents in the blood vessel tissues of thoracic aorta and mesenteric artery as well as in the plasma, [2]to measure preproendothelin mRNA levels in these blood vessels using RT-PCR and [3]to examine Kd and Bmax values of ET-1 receptor in the blood vessels using radioligand binding experiment and Scatchard plot analysis. These parameters together should give a good indication of the paracrine effects of ET-1 in the vasculature of diabetic rats.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
This investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1985). Male Sprague-Dawley rats weighing 250–300 g were divided into 3 groups: one control and two diabetic groups (one treated with insulin, one not). Diabetes was induced by intraperitoneal injection of streptozotocin (STZ, 65 mg/ Kg body weight), whereas the control animal received the buffered vehicle (0.1 mol/l citrate, pH 4.5). Induction of diabetes was confirmed by the presence of polyuria, polydipsia and glycouria in the rats. Later it was further confirmed by serum glucose measurement. Half of the diabetic rats (insulin replacement group) were given insulin in the afternoon (4 units/day) subcutaneously, 1 week after STZ injection. Control and diabetic rats received saline injection only. Two weeks or four weeks after the injection of STZ, the rats were killed in the morning by decapitation. The thoracic aorta and the mesenteric artery were quickly excised, frozen on dry ice and stored at –70°C. The trunk blood was collected into pre-chilled plastic test tubes containing 100 µl 7.2% EDTA. The blood samples were centrifuged for 20 min (2,000xg) and the plasma was aspirated, snap-frozen and stored at –70°C until extraction.

2.1 Extraction of endothelin-1 from tissues and plasma
Tissues were homogenized using a polytron in 2 N acetic acid and boiled for 10 min. The homogenate was centrifuged for 20 min at 17,000xg at 4°C. The supernatant was lyophilized and stored at – 20°C until assay. Plasma ET-1 was extracted by Sep-column according to the method described previously [20]. The extract was further purified by a Nanospin plus column (Gelman, USA, MW cut off, 5K).

2.2 ET-1 radioimmunoassay
ET-1 was measured using a specific radioimmunoassay. Duplicate samples of ET-1 standard (0–500 pg/100 µl) and the extracted samples were incubated for 18 h at 4°C with 100 µl of ET-1 antiserum (Peninsula, CA, USA) and 100 µl of 125I-labeled ET-1 (8000 cpm) prepared in our laboratory. Separation of antibody-bound from free 125I-ET-1 was achieved using goat-anti-rabbit antibody.

2.3 Extraction of tissue total RNA
Total RNA was extracted using TRIZOL reagent (GIBCOL-BRL). Thoracic aorta and the mesenteric artery were homogenized in 1 ml TRIZOL reagent using a polytron. No attempt was made to separate the smooth muscles and the endothelium. The homogenate was incubated at room temperature for 5 min; 0.2 ml of chloroform was added, and the contents were mixed by shaking the tubes vigorously for 15 s and then incubated at room temperature for 3 min. The samples were centrifuged for 15 min at 10,000xg at 4°C. The aqueous phase was collected and mixed with an equal volume of isopropanol. The RNA was precipitated at room temperature for 10 min and centrifuged for 10 min at 10,000xg at 4°C. The RNA pellet was washed with 1 ml 70% ethanol and centrifuged. The pellet was air-dried and dissolved in TE buffer (pH 7.5) and stored at –70°C until assay.

2.4 Reverse transcriptase-polymerase chain reaction (RT-PCR)
Total RNA (1 µg) from thoracic aorta and mesenteric artery was used in a RT reaction. PCR was performed with rat preproendothelin specific oligonucleotide primers. Preproendothelin primer 1 (antisense) was defined by bases 341–360, sequences 5'-TGCTCCTGCTCCTCCTTGAT-3'; primer 2 (sense), bases 792–811, sequence 5'-CACCACGGGGCTCTGTAGTC-3' [21]. The cDNA amplification product was predicted to be 471 bp in length. We performed RT-PCR of β-actin as an internal standard. Beta-actin primer 1 (antisense), bases 3055–3079, sequence 5'-ACCTTCAACACCCCAGCCATGTACG-3'; primer 2 (sense), bases 2170–2194, sequence 5'-CTGATCCACATCTGCTGGAAGGTGG-3'. Beta-actin primers spanned two introns and resulted in a 703 bp product. Taq DNA polymerase (5.0 U) was used in each PCR reaction. Ten picomoles of endothelin primers 1 and 2 were added to the reaction tubes first and after 10 cycles, 10 picomoles of actin primer 1 and 2 were added to the reaction tubes. The reaction mixture (100 µl) was overlaid with 100 µl mineral oil. PCR was programmed using a temperature control system to perform initial melt by 94°C, 3 min and 35 (for endothelin) or 25 (for actin) cycles of the following sequential steps: 94°C, 1 min (melt); 60°C, 1 min (anneal); 72°C 1 min (extend) and final extension by 72°C, 10 min. Samples were kept at –20°C until Southern blot analysis.

2.5 Southern blot analysis of PCR products
The PCR products were size-fractionated by 1% agarose gel electrophoresis together with DNA size markers ({emptyset}X174 RF DNA/Hae III fragments) ranging 72–1353 bp. DNA bands visualized by ethidium bromide staining were photographed. Then gels were blotted onto nylon membrane in 10xSSC solution. The blotted DNA was fixed using an UV-cross linker and then the membranes were baked at 80°C for 2 h. Oligoprobes (antisense DNA) positioned between 5' and 3' primers were synthesized to hybridize the PCR products. The sequence of Preproendothelin oligoprobe was 5'-CAAAGAACTCCGAGCCCAAA-3' and the sequence of actin oligoprobe was 5'-CTGCGTCTGGACCTGGCTGGCCGGG-3'. The synthetic oligonucleotide for hybridization was end-labeled with [{gamma}-32P] ATP (3000 Ci/mmol, Amersham, UK) using a 5'-end oligonucleotide labeling kit (Promega USA).Prehybridization (3 h) and hybridization (overnight) were carried out at 50°C. After the hybridization, the membrane was washed in 2xSSC, 0.5% SDS, 3 times for 10 min each, and then processed for autoradiography. The intensities of the signals from each band were analyzed using a Phosphor-imager and then the membranes were exposed to X-ray film.

2.6 Receptor binding
Thoracic aorta and mesenteric artery were homogenized using a polytron in 0.05 M Tris-HCl (pH 7.4)–0.25 M sucrose buffer. The homogenate was centrifuged at 3,000xg for 10 min at 4°C. The pellet was homogenized and centrifuged again. The supernatant was pooled and centrifuged at 100,000xg for 30 min at 4°C. The pellet was then suspended in 0.05 M Tris-HCl (pH 7.4)–0.25 M sucrose buffer containing PMSF, aprotinin, bacitracin and leupeptin. Saturation experiment was performed, briefly: 30 mg of membrane protein was incubated with different concentrations of 125I-ET-1 and with (specific binding) or without (total binding) unlabeled ET-1 (1 mM). Bound and free radioligand were separated by rapid filtration through Whatman GF/B filters. The ratio of specifically bound to free radioligand concentration is plotted against specific binding (Scatchard plot). Kd and Bmax values were then obtained from this Scatchard plot. These values are averages of the receptors in the smooth muscle cells and endothelium.

2.7 Statistics
Data are presented as mean±SE. The data were analyzed using one-way analysis of variance (ANOVA) and multiple comparison procedure was performed using Turkey's test with P<0.05 as the level of significance.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Body weights and serum glucose levels
At 2 weeks and 4 weeks after the injection of STZ, the body weights of diabetic rats were significantly lower than that of control and diabetic rats treated with insulin. Serum glucose levels of 2 week and 4 week diabetic rats were significantly higher than those of control and insulin replacement rats (Table 1). The higher serum glucose level in the insulin replacement rats than control was due to the fading effect of insulin injected on the previous afternoon.


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  		Table 1 The effects of STZ-induced diabetes on body weights (g) and serum glucose levels (mg/dl) in rats; (n=8 for all groups)

 
3.2 ET-1 levels in the plasma and in the blood vessels
Plasma ET-1 levels did not change in either 2 week or 4 week diabetic rats. However, ET-1 contents in both thoracic aorta and mesenteric artery increased significantly in 2 week diabetic rats but not in 4 week diabetic rats. Insulin replacement reversed these effects (Table 2).


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  		Table 2 ET-1 levels in the plasma (pg/ml), aorta and in the mesenteric artery (pg/mg protein) of rats injected with STZ (n=8 for all groups)

 
3.3 Preprondothelin mRNA levels in the mesenteric artery and in the thoracic aorta
Preproendothelin mRNA levels (expressed in arbitrary units after normalization to β-actin mRNA) decreased significantly in the mesenteric artery and in the thoracic aorta in 4 week diabetic rats but not in 2 week diabetic rats. Preproendothelin mRNA levels were returned to normal by insulin treatment (Table 3Fig. 1).


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  		Table 3 Preproendothelin mRNA levels (in arbitrary units) in the mesenteric artery and in the thoracic aorta (n=5 for all groups)

 

Figure 1
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  		Fig. 1 Southern blotting of the RT-PCR products of the RNAs from the mesenteric artery and thoracic aorta in the 2 week and 4 week diabetic rats. The upper bands (703 bp) are β-actin mRNA products while the lower bands (471 bp) are preproendothelin mRNA products. Control samples are in Lanes 1, 4, 7, 10 and 13; diabetic samples in Lanes 2, 5, 8, 11 and 14; and diabetic + insulin samples in Lanes 3, 6, 9, 12 and 15.

 
3.4 ET-1 receptor in the mesenteric artery and in the thoracic aorta
The Kd and Bmax of ET-1 receptor in the mesenteric artery and in the thoracic aorta increased in both 2 week and 4 week diabetic rats. Insulin group showed a slightly higher values of Bmax and Kd compared to control according to power calculation. The lack of difference in some parameters between insulin group and control group may be due to a type II error resulting from the small number of samples in each group (Table 4).


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  		Table 4 Bmax and Kd values of ET-1 receptor in the mesenteric artery and in the thoracic aorta (n=3 for all groups)

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
In this study we have found that, in STZ-diabetes, there was a decrease in ET-1 release from the thoracic aorta and the mesenteric artery and an increase in receptor binding in these tissues. We did not see any change of plasma ET-1 levels in STZ-diabetic rats nor in insulin treated rats although a decrease after 4 days [10]and an increase after 10 weeks [5]of STZ-injection have been reported. However, ET-1 content in the mesenteric artery and in the thoracic aorta increased in 2 week (but not in 4 week) diabetic rats. That this effect was reversed by insulin shows that it was due to a lack of insulin or its metabolic consequence and not due to the toxicity of STZ. The synthesis and release of ET-1 are affected by many mechanisms including humoral and mechanical factors. In vitro experiment showed that glucose in culture medium (5.5–22.2 mM) stimulated ET-1 secretion from endothelial cells [22]while higher (27.5 mM) glucose reduced ET-1 release [23]. Our diabetic rats had a serum glucose higher than 27.5 mM and it is possible that under such a high glucose condition endothelin secretion from the endothelium in vivo is also reduced. Though positive immunoreactivity of ET-1 has been reported in the vascular smooth muscle, it is a common belief that ET-1 comes mostly from the endothelial cells of the blood vessels [1].

The preproendothelin mRNA results indicate that there was an increase in endothelin synthesis in the thoracic aorta and mesenteric artery in the 4 week STZ-diabetic rats but not in the 2 week STZ-diabetic rats. This, together with the ET-1 results, suggests that ET-1 release was decreased in both the 2 week (no change in mRNA, increase in peptide) and 4 week (decrease in mRNA, no change in peptide) STZ-diabetic rats. Hence in STZ-diabetic rats, the decrease in the release of ET-1 from the endothelium preceded a decrease of ET-1 synthesis. At any rate, a decrease in ET-1 release would lead to a decrease in local concentration in vascular smooth muscle. It is interesting to note that Takeda et al. has reported an increase in ET-1 release from perfused mesenteric artery of diabetic rats 10 weeks after STZ-injection compared with control rats [5]. How far this in vitro result can be extrapolated to the in vivo situation is open to question.

The finding of an increase in the Kd and Bmax of endothelin receptors (most probably ETA receptors) in the blood vessels in both 2 week and 4 week diabetic rats, which is in line with a decrease in ET-1 release, also supports our speculation. Data in this aspect are lacking in the literature [24]. Though ETB receptor subtype is present in some vascular preparations [25, 26], there is also evidence suggesting that ETB receptors are not present in the smooth muscle preparation from the rat. It is generally accepted that in most vascular smooth muscle preparations, ET-1 interacts with a specific cell surface receptor, the ETA receptor subtype. We could not study the ET receptor subtypes because of the availability of the tissue. Given the above results, it is suggested that the release of ET-1 from the endothelium is decreased in STZ-induced diabetes and results in the up-regulation of ET-1 receptor in the vascular smooth muscle. This is in agreement with the finding of Kiff et al. [27]of an increase in vasoconstrictive response to ET in 4 week diabetic rats. It is at variance with the results of Fulton et al. [14]and Hodgson and King [15]of a decreased reactivity to ET in the aorta in 2 week and 6 week diabetic rats and suggests that the decrease in reactivity observed may be a post-receptor event. The possibility that similar change of ET-1 receptor occurred in diabetic rats treated with insulin cannot be entirely excluded. On the other hand, our RT-PCR results indicate that the preproendothelin mRNA levels in the mesenteric artery and in the thoracic aorta decreased in 4 week but not in 2 week diabetic rats, supporting our speculation that the synthesis of ET-1 in the blood vessels is progressively impaired in STZ-induced diabetic rats. However, it should be noted there is a limitation of the use of STZ-diabetic rats in relation to human diabetes in that although STZ acts by destroying β-cells that produce insulin, it may have some non-specific effects unknown to us.

Time for primary review 21 days.


   Acknowledgements
 
This work was supported by a grant from the Committee on Research and Conference Grant (335/034/0072).


   Notes
 
1 Present address: Department of Physiology, Beijing Medical University, Beijing 100083, China. Back


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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S.-Q. Wu, R. L Hopfner, J.R. McNeill, T. W Wilson, and V. Gopalakrishnan
Altered paracrine effect of endothelin in blood vessels of the hyperinsulinemic, insulin resistant obese Zucker rat
Cardiovasc Res, March 1, 2000; 45(4): 994 - 1000.
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