HYPERTENSIVE DIABETIC RATS IN PHARMACOLOGICAL STUDIES

Pharmacological Research, Vol. 33, No. 2, 1996

HYPERTENSIVE DIABETIC RATS IN PHARMACOLOGICAL STUDIES P. A. VAN ZWIETEN, K. L. KAM, A. J. PIJL, M. G. C. HENDRIKS, O. H. M. BEENEN and M. PFAFFENDORF Departments of Pharmacotherapy and Cardiology, Academic Medical Centre, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands Accepted 23 May 1996 Since hypertensive disease and diabetes frequently occur simultaneously there exists a requirement for animal models where both pathological entities are combined. The streptozotocin (STZ)-spontaneously hypertensive rat (STZ-SHR) and the obese Zucker rat are examples of animal models where hypertension and diabetes occur simultaneously. STZ-SHRs develop a hyperglycaemic syndrome, associated with other biochemical and morphological changes that to some extent approach insulin-dependent diabetes mellitus (type 1 diabetes) combined with hypertension. The obese (Fa/?) Zucker rat is characterized by the simultaneous occurrence of obesity, hyperglycaemia, hyperinsulinaemia, hyperlipidaemia and moderate hypertension. As such it approaches the patient with non-insulin-dependent diabetes mellitus (type 2 diabetes) who is simultaneously hypertensive. Lean (fa/fa) Zucker rats are suitable controls with respect to the obese animals. Both animal models (STZ-SHRs and obese Zucker rats) were characterized with respect to their biochemical, morphometric and haemodynamic properties. Both models were examined in particular with respect to the pharmacological characteristics of their cardiovascular system, as discussed in the present survey.  1996 The Italian Pharmacological Society KEY WORDS: hypertension, diabetes mellitus, streptozotocin hypertensive rats, obese Zucker rats, cardiovascular system.

INTRODUCTION Hypertensive disease and diabetes mellitus are known to act synergistically with respect to the cardiovascular damage associated with both disorders. Since both conditions frequently occur simultaneously much interest has developed in the fields of hypertension and diabetes research concerning the backgrounds, relevance and sequelae of their combined occurrence, both in animal models and in patients. Hypertensive disease is mimicked in several animal models. It may be acquired on the basis of perinephritis, DOCA salt exposure, etc., or develop on a genetic basis, as in the well-known spontaneously hypertensive rat (SHR), of which various subtypes are available at present. For review see [1–3]. In addition, diabetic animals are now also available for research purposes. Experimental diabetes resembling insulin-dependent diabetes mellitus (IDDM; type 1 diabetes) can be induced in rodents, for instance by means of alloxan or, preferably, streptozocin (STZ). An IDDM-like syndrome is observed in animal strains which spontaneously develop diabetes, such as NOD mice, BB rats, LETL rats, Chinese hamsters and Keeshond dogs. Non-insulin-dependent diabetes mellitus (NIDDM) (type 2) syndromes in 1043–6618/96/020095–11/$18.00/0

animals occur predominantly on a genetic basis. A few examples of such animals are db/db mouse

obese, severe diabetes

ob/ob mouse New Zealand obese mouse obese Zucker rat WKY fatty rat

obese, mild diabetes

Swiss-Hauschka mouse Cohen diabetic rat

diabetes developed by selective inbreeding

An NIDDM-like syndrome can also be induced by nutrition, such as in the sand rat, spiny mouse and Mongolian gerbil. For reviews on animal models of diabetes see [4–6]. The aforementioned, frequently occurring combination of hypertensive and diabetic disease has led to the search for animal models approaching this complex condition. Accordingly, SHRs can be rendered diabetic by means of a single injection of STZ. This model (STZ-SHR) was developed a few years ago, and predominantly used for fundamental, biochemical studies in diabetes research [7, 8]. The model is easy to handle, reproducible and suitable for pharmacological and biochemical investigations of diabetes and 1996 The Italian Pharmacological Society

96

STREPTOZOCIN-INDUCED DIABETIC SHR For detailed descriptions see [13–16]. SHRs (male, body mass 200–250 g, 12 weeks old) received a bolus injection of STZ (55–60 mg kg−1) in a lateral tail vein. The animals were kept for 8 weeks after the STZ injection and then used for pharmacological experiments. Male WKY rats, treated according to the same protocol, were used as controls. The time course of the rise in plasma glucose levels in the STZ-treated animals is visualized in Fig. 1. Already 3 days after the injection of STZ, the plasma glucose levels of both types of animals proved significantly elevated to a diabetic level, and body weights were significantly reduced. The STZ-SHR showed a stronger weight loss than the STZ-WKY. These differences persisted over 8 weeks, just prior to the pharmacological experiments to be described in forthcoming sections. In both groups of diabetic animals the fur proved discoloured and the skin showed erythema. Other typical symptoms were polyuria, dehydration, albuminuria, glucosuria, diarrhoea, a swollen intestine (in

(A)

*

*

*

*

*

–1

Plasma glucose (mmol l )

30

20

*

10

0 0 3

28 Time (days)

*

*

56

400 (B) 350 Body mass (g)

hypertension, combined as well as separated. One of its major drawbacks, however, is the fact that STZinduced diabetes rather resembles type 1 than type 2 diabetes, whereas in the human situation the simultaneous occurrence of both diseases more frequently concerns type 2 diabetes. Conversely, the obese Zucker rat, first described in 1965 [9] as a result of cross-breeding of Sherman and Merck Stock M rats, develops the following symptoms resembling the NIDDM syndrome: a. obesity, as a result of a positive energy balance induced by both hyperphagia and low physical activity; b. insulin resistance, hyperinsulinaemia and glucose intolerance; c. hyperlipidaemia; d. arterial hypertension. Because of the simultaneous occurrence of diabetes and moderately elevated blood pressure, these rats, now commercially available, may also be considered as hypertensive diabetic rats. Similarly to the STZSHR the obese Zucker rats have been subjected predominantly to fundamental studies in diabetes research. For reviews see [9–12]. So far both types of hypertensive diabetic rats have not been used on a large scale in detailed pharmacological studies, although they would appear as potentially useful models to investigate pathopharmacological changes in the organs and tissues associated with the combination of both diseases. Accordingly, our group has further characterized both STZ-SHR and obese Zucker rats in more detail, and used both types of hypertensive diabetic animals for a series of pharmacological investigations as described in the present survey.

Pharmacological Research, Vol. 33, No. 2, 1996

300 *

250 * 200 0 3

* 28 Time (days)

* 56

Fig. 1. (A) Mean plasma glucose levels (mmol l−1 ) and (B) body mass (g) established at several times (days) just prior and after the injection of STZ (day 0) or vehicle in the four groups of rats: STZ-induced diabetic normotensive (STZWKY) and hypertensive (STZ-SHR) rats and age-matched control rats (WKY and SHR), respectively. Data represent means±SEM, n$28, *P<0.05 vs control (WKY or SHR); †, P<0.05 vs WKY (STZ or control); s, control WKY; d, STZ-WKY; h, control SHR; j, STZ-SHR.

particular the caecum), and a pronounced loss of abdominal adipose tissue. In spite of the obviously sick appearance of the diabetic animals the mortality rates over 8 weeks were similar in the four groups of animals: STZ-WKY, 2.7%; STZ-SHR, 2.7%; non-diabetic WKY, 3.4%; non-diabetic SHR, 0.7%. The diabetic animals did not receive insulin treatment. It is well known that such a treatment of STZ-hyperglycaemic rats is not required for their survival for this period of 8 weeks. Analysis of the blood gases and acid–base status of conscious rats of the four categories may be characterized as follows: pH, p CO2, pO2, O 2 saturation and [HCO3−] were found to be in the normal range in all four categories of animals; however, the base excess in the diabetic animals (both WKY and SHR) proved above normal (>2). Analysis of the urine revealed, as to be expected, a markedly elevated glucose level in both types of diabetic animals (Table I). A higher protein level, when compared with the diabetic rats, was found in both normoglycaemic WKY and SHR (Table I). Ketone bodies were found in the urine and blood of hypergly-

Pharmacological Research, Vol. 33, No. 2, 1996

97

caemic animals, and their levels were higher in STZSHR than in STZ-WKY. In normoglycaemic animals no ketone bodies could be detected in the urine or blood.

diabetes. It seems likely that both STZ-induced diabetes and hypertension may increase the hypertrophy of the arteries up to a certain maximal level, as indicated by our finding that the tunica media thickness of small arteries taken from STZ-SHR was not different from that in control SHR vessels.

Morphometric (histological) characteristics of the cardiovascular system in STZ-SHR

The heart

Small arteries. As summarized in Table II the tunica media in small mesenteric arteries was significantly increased in the control hypertensive group (control SHR vs control WKY), but not in the preparations from the diabetic hypertensive group (STZSHR vs STZ-WKY). The induction of STZ diabetes led to a significantly increased thickness of the tunica media for the normotensive group (STZ-WKY vs control SKY). However, this difference was not found in preparations from both groups of hypertensive animals (STZ-SHR vs control SHR). The calculated tunica media thickness to lumen ratio was increased in SHR preparations when compared with those taken from control WKY. The changes in tunica media thickness as a result of hypertension reflect vascular hypertrophy associated with the hypertensive state. This finding has been described several times before [17–19]. Very little has been reported so far concerning the vascular morphology in a condition of STZ-induced

Ventricular wall thickness. Measurements of ventricular wall thickness for each of the four groups of hearts are summarized in Table III. Both diabetic hearts (diabetic WKY and diabetic SHR) showed a significantly (P<0.05) thinner left ventricular wall compared with the normoglycaemic WKY and SHR. There were no differences in right ventricular wall thickness between the four groups. Ventricular septum thickness was significantly (P<0.05) thinner in diabetic SHR vs normoglycaemic SHR, but not in diabetic WKY vs normoglycaemic WKY. Left ventricular wall to lumen ratio. This was marginally significantly decreased in hearts from diabetic WKY and diabetic SHR when compared with their normoglycaemic counterparts. This ratio was significantly decreased in diabetic SHR when compared with the WKY. Histopathological findings. Alterations in myo-

Table I Glucose and protein levels of urine, and of ketone bodies in blood and urine derived from WKY, diabetic WKY, SHR and diabetic SHR Group

Glucose in urine,

Protein in urine,

Ketone bodies in urine,

Ketone bodies in blood,

score/n (0–1–2–3–4)

score/n (0–1–2–3–4)

score/n (0–1–2–3)

score/n (0–1–2–3)

WKY

0.44 (n=25)

2.72 (n=25)

0.00 (n=5)

0.00 (n=4)

Diabetic WKY

4.00 (n=18)

1.24 (n=25)

0.19 (n=21)

0.41 (n=22)

SHR

0.00 (n=18)

2.74 (n=27)

0.00 (n=5)

0.00 (n=5)

Diabetic SHR

3.88 (n=16)

2.22 (n=18)

1.26 (n=19)

1.10 (n=20)

The data are presented as scores divided by n. The score scale for each parameter is given in parentheses. For abbreviations see text.

Table II Morphological characteristics of isolated mesenteric small arteries taken from STZ WKY, STZ SHR and appropriate age-matched control rats Tunica media thickness ( µ m) Tunica media thickness to lumen diameter ratio (%)

Control WKY

STZ WKY

Control SHR

STZ SHR

14.42±0.59

18.45±1.11*

21.31±0.63†

19.60±0.87

5.31±0.33

6.34±0.43

6.71±0.39†

5.94±0.43

The preparations were fixed within the myograph at normalized equilibrium baselines and embedded later in Epon for the assessment of the tunica media thickness, and tunica media thickness to lumen diameter ratio. For abbreviations see text. Data are presented as means± SEM, n=5, except for control WKY, n=4; *P<0.05 vs control (WKY or SHR), †, P<0.05 vs SKY (STZ or control).

98

Pharmacological Research, Vol. 33, No. 2, 1996

(a)

(a)

(b) (b)

Fig. 3. Section of the myocardium of (a) a WKY and (b) an SHR. Haematoxylin–eosin stain; ×50.

Fig. 2. Section of the heart (a) an SHR and (b) a diabetic SHR, showing both ventricles at the midlevel of the papillary muscles. Haematoxylin–eosin stain; ×50.

cardial histology could be found in the normoglycaemic and diabetic SHR, but not in the normoglycaemic and diabetic WKY. Myocardial hypertrophy, characterized by an increase in cardiomyocyte size

and relative reduction in the number of nuclei (myocytes and interstitial cells per high power field (HPF)) was observed in the normoglycaemic SHR only (Fig. 2,3). With respect to the extracellular matrix, an increase in perivascular collagen content was seen in both the normoglycaemic and the diabetic SHR (Fig. 3). The pattern of interstitial collagen distribution was similar in all four groups. However,

Table III Quantification of histological parameters (means6 SEM) established in hearts from WKY, diabetic WKY, SHR and diabetic SHR n

Group

LV wall

RV wall

Septum

(mm)

(mm)

(mm)

Wall: lumen

Media

Collagen content

3

WKY

3.09±0.14

0.88±0.07

2.97±0.15

6.91±0.62

0.09±0.00

0.056±0.003

3

Diabetic WKY

2.24±0.09*

0.97±0.14

2.23±0.36

4.40±0.82*m

0.10±0.00*

0.075±0.010

3

SHR

3.40±0.07

0.88±0.00

3.27±0.25

8.46±1.44

0.14±0.02#µ

0.047±0.002

3

Diabetic SHR

2.59±0.13*

0.77±0.11

2.32±0.17*

4.54±0.58*m

0.15±0.02#µ

0.056±0.004

Shown are the thickness of the left ventricular (LV) wall, the right ventricular (RV) wall, the interventricular septum and the left ventricular wall to lumen ratio. The media of the coronary arteries and the collagen content were calculated as a ratio. *means significantly (P<0.05) different from the corresponding normoglycaemic group; #means significantly (P<0.05) different from the corresponding normotensive group; mmeans marginally significant (0.0651
Pharmacological Research, Vol. 33, No. 2, 1996

99

quantitative collagen content analysis showed that the perivascular fibrosis in hypertensive rats did not significantly affect the total collagen content of the myocardium, which was the same in all groups. Vascular changes consisted of medial hypertrophy of the intramyocardial arteries in both groups of SHR. There was no intimal hyperplasia in these vessels. Moreover, no obvious changes in capillary vessels or veins could be found. The decrease in heart weight in diabetic animals, as established after dehydration by means of freeze drying, was caused by the loss of myocardial tissue and not water, since the water content of the hearts was the same (<77%) in the four groups of animals studied. In brief, hypertension is accompanied by cardiac hypertrophy, whereas the diabetic state is associated with left ventricular wall dilatation. These findings are largely in agreement with earlier reports in the literature [20]. The STZ-SHR model shows important similarities in the combined effects of diabetes and hypertension in humans with respect to cardiac remodelling (hypertrophy and dilatation) [21]. In contrast to other groups [22] who studied renovascular hypertension in rats we did not observe signs of fibrosis or myocytolysis. In our experiments both diabetes and hypertension caused medial hypertrophy, although the differences between preparations from hypertensive and normotensive animals were only marginally significant.

Haemodynamic characteristics of streptozotocindiabetic hypertensive rats As to be expected, both the hyperglycaemic and the euglycaemic hypertensive animals (STZ-SHR and control SHR) had higher blood pressure parameters (systolic, diastolic and mean arterial pressure) and heart rates compared with their normotensive controls (STZ-WKY, control WKY). The induction of STZ diabetes in the normotensive animals raised blood pressure values, but appeared to reduce heart rate. In the hypertensive rats, however, both blood pressure and heart rate were significantly reduced by the induction of STZ diabetes. Others [23, 24] also observed that the induction of STZ diabetes in SHR was associated with a lowering of blood pressure values, possibly as a result of hypovolaemia, provoked by osmotic diuresis. Other studies performed in STZ-diabetic normotensive rats show incon-

sistent results regarding blood pressure values [25]. Although the reasons for these discrepancies are unknown, they appear to be unrelated to differences in the dose of STZ, time course after treatment, or the strains of rats used. Isolated hearts perfused according to Langendorff showed an impaired contractile performance (as reflected by left ventricular pressure and dP/dtmax) when obtained from STZ-diabetic animals. In addition, coronary flow in these preparations proved reduced (Table IV). The same changes were found in hearts from diabetic SHR, indicating that it was the diabetic and not the hypertensive state which had caused these alterations. Similarly, the contractility in isolated working heart preparations was lower in the hearts of diabetic SHR when compared with non-diabetic WKY preparations (Table IV). These findings appear to be in accordance with literature data [25].

THE OBESE ZUCKER RAT In our own experiments obese Zucker rats were compared with their lean counterparts as controls. Animals from the obese strain gained weight more rapidly than the lean controls. From 9 weeks of age onwards the obese rats had a significantly higher body weight than the lean controls. The obese animals gained body weight as a result of an increase in adipose tissue, in particular in the abdominal region (Table V). Plasma levels of glucose, insulin, total cholesterol and triglycerides proved significantly higher in the obese than in the lean Zucker rats. Similarly, glucose and albumin concentrations in the urine proved elevated in the obese rats when compared with lean controls (Table V). These biochemical–metabolic data are in accordance with several reports in the literature [26–29].

Morphological characteristics In a light-microscopic investigation of small mesenteric arteries, which may be considered as resistance vessels, we found no difference between preparations taken from the two categories of rats (obese vs lean), regarding the following parameters: tunica media thickness; tunica media thickness to lumen ratio. Accordingly, the aforementioned metabolic changes

Table IV Characteristics of the isolated perfused hearts, obtained from the different categories of rats investigated (for details see text and Table I) LVP (mmHg) +dp/dtmax (mmHg s−1 ) −dp/dtmax (mmHg s−1 ) CF (ml min−1 g −1)

Control WKY

n

Diabetic WKY

n

Control SHR

n

Diabetic SHR

n

87.6±3.8 3055±136 −1912±76 8.9±0.2

24 24 24 24

48.0±2.7* 1506±102* −977±67* 6.9±0.2*

20 20 20 18

85.0±3.1 3050±139 −1910±69 8.1±0.2#

20 20 20 20

48.9±2.7* 1514±97* −880±62* 6.6±0.2*

18 18 18 18

LVP, left ventricular pressure; +dP/dt max , maximally developed contraction velocity; dP/dtmax, maximally developed relaxation velocity; CF, coronary flow expressed as ml min−1 (g wet heart mass)−1 .

Pharmacological Research, Vol. 33, No. 2, 1996

and the mild degree of hypertension (following section) do not lead to visible vascular damage during the period of observation of 22 weeks.

Haemodynamic characteristics of obese Zucker rats As shown in Table V all blood pressure parameters (systolic, diastolic, mean arterial pressure; amplitude) were moderately but significantly higher in obese than in lean Zucker rats. However, heart rate proved significantly lower in the obese than in the lean rats. In conclusion, the obese Zucker rat suffers from the syndrome of obesity–hypertension–insulin resistance–hyperlipoproteinaemia. At least in the period of observation of our animals (22 weeks) structural changes in vascular smooth muscle could not be detected, in contrast to STZ-SHR. However, such changes might occur in obese Zucker rats when observed for longer periods. The obese Zucker rat appears to be an attractive model for NIDDM (type 2) diabetes and simultaneous hypertension, as in humans.

PHARMACOLOGICAL EXPERIMENTS: STZSHR

Isolated blood vessels–vascular beds The isolated perfused mesenteric vascular bed is considered to consist predominantly of resistance vessels (precapillary arterioles). The basal haemodyTable V General characteristics of lean and obese Zucker rats at an age of 22 weeks Lean Zucker rat

Obese Zucker rat

Body mass (g) 407.2±5.2 Plasma glucose (mmol l−1 ) 6.2±0.2 Insulin (ml U l−1 ) 15.6±2.5 Cholesterol (mmol l−1 ) 1.78±0.06 HDL (mmol l−1 ) 1.12±0.06 LDL (mmol l−1 ) 0.41±0.08 Triglycerides (mmol l −1) 0.54±0.09 Urinary albumin (scale 0–4) 2.1±0.1 normal Urinary glucose (mmol l− 1)

587.0±6.8* 11.0±0.8* 59.7±4.0* 4.28±0.41* 2.83±0.39* 0.93±0.17* 1.18±0.17* 3.0±0.1* 1.6±0.6*

Systolic BP (mmHg) Diastolic BP (mmHg) MAP (mmHg) Amplitude (mmHg) Heart rate (beats min−1 )

179.8±3.2* 113.3±2.3* 135.5±2.3* 68.2±2.2* 329±5*

152.1±2.7 106.5±1.7 121.7±2.0 45.6±1.5 346±7

Shown are the mean body mass, plasma levels of glucose and insulin, and serum levels of cholesterol, HDL, LDL and triglycerides, urinary concentrations of albumin and glucose. Haemodynamic parameters are systolic, diastolic, and mean arterial blood pressures (MAP=(systolic+3× diastolic)/3), pressure amplitude and heart rate values. Data are presented as means± SEM, n=35, except for insulin and serum lipid levels, n=7; *, P<0.05 vs lean Zucker rat.

Perfusion pressure (mmHg)

100

100 75 50 25 0 –9

–8

–7 –6 –5 log [methacholine] (M)

–4

Fig. 4. Concentration–response curves for the dilator responses to the endothelium-dependent muscarinic agent metacholine in the perfused mesenteric vascular bed preparation taken from normotensive WKY (s) and SHR (h), age-matched diabetic WKY (d) and age matched diabetic SHR (j), respectively. Vascular preparations were precontracted to a resistance to perfusion of approximately 100 mmHg with equieffective concentrations of methoxamine. Data represent means±SEM (n=6). Note the blunted vasodilator response to metacholine in the preparations from diabetic rats. Similar reductions in response were found for histamine and adenosine, but not for sodium nitroprusside (data not shown).

namic characteristics of such preparations were the same in preparations obtained from non-diabetic WKY, non-diabetic SHR, diabetic WKY and STZSHR. Vasoconstrictor and -dilator responses in such preparations are quantified by means of changes to resistance to perfusion. The hypertensive state was associated with an enhanced vasoconstrictor response to the α 1-adrenoceptor agonist methoxamine. The phenomenon has been observed repeatedly for various types of vasoconstrictor agents, in isolated vessels, animal models and hypertensive patients [30–33], probably as a result of vascular structural changes (hypertrophy) which are characteristics for hypertension. The induction of the diabetic state by STZ clearly blunted this enhanced constrictor response. The diabetic state, in both normotensive and hypertensive animals (STZ-SHR) decreased the sensitivity (in the mesenteric bed preparations) with respect to the responses to endothelium-dependent vasodilator agents (metacholine, histamine, adenosine diphosphate) (see Fig. 4). By contrast the response to the endothelium-independent vasodilator sodium nitropruside proved unchanged in preparations obtained from diabetic animals (both WKY and SHR). These findings are indicative of a diabetes-induced endothelial dysfunction in the mesenteric artery preparation. In preparations from diabetic hypertensive rats the reduced response to methoxamine and the endothelial dysfunction seem to run parallel to each other. Additional experiments were performed in isolated mesenteric arteries by means of an isometric myograph (Mulvany technique). The size of the vessels

Pharmacological Research, Vol. 33, No. 2, 1996

Isolated hearts In isolated hearts perfused according to Langendorff we investigated the inotropic response to various agents with different modes of action. The hearts of diabetic rats (normotensive) showed a tendency (non-significant) towards a somewhat impaired inotropic response to extracellular calcium ions, and this effect seemed to be enhanced by hypertension of the donor animals. However, the inotropic response to the calcium entry promoter Bay k 8644 proved the same in the isolated hearts taken from all four categories of rats (WKY, diabetic WKY, SHR, STZ-SHR). This finding indicates that there are probably no important differences in the properties of the cardiac L-type calcium channels in the diseased state [36]. The inotropic responses to both cirazoline and ST 587 were increased in hearts from diabetic WKY when compared with those from control WKY. The hypertensive state induced a blunting of the inotropic responses to ST 587 in hearts from control SHR and diabetic SHR, but not to cirazoline [36]. The interaction with calcium handling by these two compounds is known to be fundamentally different, as described previously [37]. The difference in response to cirazoline and ST 587 may indicate that the calcium influx in the hypertensive state is impaired since the hypertensive state only influenced the response to ST 587. Our finding that the responses to both agents were

* 80 LVP (recovery %)

was such that they can be considered as resistance vessels. Again the preparations were obtained from non-diabetic WKY, non-diabetic SHR, diabetic WKY and STZ-SHR. The vasoconstriction caused by α1adrenoceptor stimulation (methoxamine, noradrenaline) and serotonin proved to be influenced by neither the hypertensive nor the diabetic state [34]. STZ-induced diabetes (in both normotensive and hypertensive rats) reduced the sensitivity (as reflected by −log EC 50 values) of the preparations for CaCl2 induced vasconstriction. Similar findings were obtained with respect to the vasoconstriction induced by a depolarizing KCl solution [34]. In conclusion, the STZ-diabetic state did not cause pharmacodynamic changes, neither in preparations taken from STZ-WKY nor in those from STZ-SHR, in spite of the morphological alterations found in such vessels. Similar experiments were performed under the same conditions (isolated vessels; isometric myograph) with various types of vasodilator agents. The isolated vessels had been precontracted by means of methoxamine. STZ-induced diabetes proved to enhance the responses to histamine, bradykinin, and cromakalim, whereas those to sodium nitroprusside, metacholine and nifedipine were unchanged [35]. These findings suggest that the influence of the diabetic state is more pronounced than that of hypertension. In addition these data do not indicate that either hypertension or diabetes is associated with generalized endothelial damage in the resistance vessels.

101

Nifedipine Control *

60 40 20 0

SHR WKY Diabetic WKY Time (days)

Diabetic SHR

Fig. 5. The recovery of the left ventricular pressure (LVP) in isolated working heart preparations after 30 min of lowflow ischaemia, after treatment with nifedipine in the EC 60 concentration; WKY (2.3×10 −7 M), diabetic WKY (1.3± 10−7 M), SHR (1.6±10 −7 M), and STZ-SHR (1.5±10−7 M ). Data: means±SEM (n=48). Control experiments with the vehicle. Values marked with an asterisk (*) are significantly higher than the control values.

enhanced indicates that the diabetic state would influence both the cellular calcium inward flux and intracellular processes [36]. The present study indicates that the combination of hypertension and diabetes lead to progressive cardiac deterioration, as observed in previous experiments. This deterioration is likely to be the result of important changes in calcium homeostasis. The pharmacodynamic changes observed in our experiments also point towards relevant changes in calcium handling by the myocardium. Accordingly, the influence of hypertension and diabetes on the contractile responses to α 1adrenoceptor stimulants and to calcium ions reflects substantial changes in calcium handling in such hearts. In a second series of experiments we set up isolated working heart preparations of STZ-SHR and compared these with appropriate controls. The dihydropyridine-calcium antagonist nifedipine, as to be expected, significantly depressed cardiac contractile force, as reflected by reductions in left ventricular pressure, dP/dtmax , aortic output and cardiac output. The effect of nifedipine was approximately the same in preparations obtained from WKY, diabetic WKY, SHR, and STZ-SHR [38]. In connection with a programme of investigations on experimental myocardial ischaemia, isolated hearts were subjected to low flow ischaemia, which led to important reductions in contractile force and coronary flow. Nifedipine did not influence the recovery of these parameters in hearts from non-diabetic rats, but a protective effect of nifedipine against the sequelae of ischaemia was found in hearts from both diabetic WKY and SHR [38] (Fig. 5). Similarly, the experimental anti-ischaemic agent R 56865 proved also protective in hearts from diabetic WKY and SHR recovering from low-flow ischaemia

Change diastolic pressure (mmHg)

102

Pharmacological Research, Vol. 33, No. 2, 1996

effects of all agents studied [40]. This phenomenon is well known in various animal models as well as in hypertensive patients. It is unlikely to be caused by receptor changes, but rather reflects the vascular hypertrophy known to be associated with hypertensive disease [30–33]. Interestingly, STZ-induced diabetes caused an opposite effect: the various pressor effects were clearly blunted in diabetic animals.

150

100

50

0

–9

–8 –7 –6 log [ST587] (mol kg–1)

–5

–4

Fig. 6. Increase in diastolic blood pressure induced by intravenous administration of the α 1 -adrenoceptor agonist ST 587 in four categories of pithed rats: control SKY (h), diabetic WKY (j), control SHR (s), and diabetic SHR (d). Symbols represent mean± SEM (n=5 or 6). Note the blunted pressor responses in diabetic rats, which is opposite to the enhanced response in hypertensive rats.

[39]. This protection was also observed in hearts of normoglycaemic animals, in contrast to nifedipine. The diabetic state appears to enhance the cardioprotective activity of nifedipine and R 56865 against the sequelae of ischaemia.

Pithed rats Pithed rat preparations were made from animals that were diabetic, hypertensive, or both and compared with appropriate controls, as in the experiments with isolated vessels and hearts. We compared the vasoconstrictor pressor effects of a few α 1- or α 2-adrenoceptor agonists, and angiotension II in the aforementioned categories of animals (Fig. 6). The hypertensive state induced an enhancement of the pressor

PHARMACOLOGICAL EXPERIMENTS; OBESE ZUCKER RATS Since the obese Zucker rat has become available but recently, it has so far hardly been used for pharmacological studies. We performed a pharmacological investigation with isolated resistance vessels taken from obese and from lean Zucker rats that were 22 weeks of age. As discussed in a preceding section the obese animals were hyperglycaemic and they had moderate hypertension. The tunica media thickness and the calculated tunica media thickness to lumen diameter ratio were the same in mesenteric artery preparations taken from obese or lean Zucker rats. In isolated resistance vessels of these animals we studied the functional responses to several vasoconstrictor and dilator agents [41]. Cumulative concentration response curves (CRCs) for noradrenaline (with uptake 1, uptake 2 and β 1, β 2adrenoceptor blockade), methoxamine and serotonin were constructed. No differences were found in the CRCs with respect to the sensitivity of the preparations for noradrenaline, methoxamine and serotonin or for the slope or maximal active force [41]. Similarly the CRCs constructed for the effects of calcium chloride (added to a fully depolarizing K+-

Table VI Characteristics of the cumulative concentration–response curves for noradrenaline, methoxamine, serotonin, calcium chloride and potassium chloride (the mesenteric small arteries were isolated from lean and from obese Zucker rats) −Log EC50

Slope

Emax (mN mm−1 )

Noradrenaline

Lean Zucker rat Obese Zucker rat

5.50±0.08 5.59±0.06

1.70±0.09 1.72±0.07

3.72±0.12 3.67±0.16

Methoxamine

Lean Zucker rat Obese Zucker rat

5.75±0.04 5.67±0.17

2.79±0.39 2.68±0.22

4.04±0.14 3.70±0.25

Serotonin

Lean Zucker rat Obese Zucker rat

6.58±0.03 6.55±0.07

2.21±0.18 2.22±0.11

3.80±0.19 3.51±0.26

Calcium chloride a

Lean Zucker rat Obese Zucker rat

3.45±0.08 3.47±0.03

1.54±0.08 1.52±0.05

3.25±0.11 3.44±0.44

Potassium chlorideb

Lean Zucker rat Obese Zucker rat

1.22±0.01 1.22±0.03

3.76±0.19 3.68±0.18

2.84±0.30 2.96±0.27

Data are presented as means±SEM , n=7 (except for calcium chloride, n=6). EC 50 (mol l− 1), median effective concentration; E max (mN mm−1 ), maximal active force. aAdded to a fully depolarizing K+ -PSS (120 mmol l−1 ). b Added to a PSS containing 1.8 mmol l− 1 CaCl2 .

Pharmacological Research, Vol. 33, No. 2, 1996

Tyrode solution (120 mmol K+ l−1 )) and those for the influence of increasing potassium concentrations also proved the same for both types of preparations (Table VI). Obviously, the diabetic state of the donor animals did not lead to significant changes in sensitivity, slope or maximal active force of the CRCs as constructed for the isolated preparations [41]. The following vasodilator agents were investigated: sodium nitroprusside (endothelium independent) and metacholine (endothelium dependent). For both vasodilator agents the CRCs were the same in preparations taken from obese and from lean Zucker rats. Similarly, small arteries isolated from both lean and obese Zucker rats showed the same CRCs for the vasodilator effect of cromakalim. Pretreatment of the preparations with glibenclamide (3×10−6 mol l−1 ) shifted the CRCs of cromakalim to the right, whereas the slopes of the CRCs proved depressed. However, CRCs constructed for the preparations taken from both categories of rats were the same. Nifedipine inhibited the tonic component of the K+-induced contractions to the same extent (log IC50 (mol l−1), slope and maximal inhibition (per cent)) for the preparations taken from both types of rats [41]. The inhibitory effect of nifedipine on the phasic response of K+-induced contractions was established as well. The sensitivities, slopes and the maximal inhibition of this phasic response (percentage from the initial value) were not different for the isolated vessels (values for lean Zucker rat: 7.69±0.16 mol l−1 (−log IC 50), −1.65±0.34 (slope) and 54.53±6.24% (maximal inhibition)) [41]. In conclusion, the various vasoconstrictor and dilator agents caused the same effects in resistance arteries obtained from either obese or lean Zucker rats. Our observations are in agreement with those found by Cox and Kikta [42] who observed no significant differences in concentration–response relations (of thoracic aorta preparations isolated from obese Zucker rats) for acetylcholine and sodium nitroprusside. However, Auget et al. [43] observed that the presence of the endothelium counteracted the concentration induced by phenylephrine more clearly in vessels from obese than in those obtained from lean Zucker rats. An impaired smooth muscle relaxation response to acetylcholine and glyceryltrinitrate was observed in aortae from obese Zucker rats [44]. In the obese Zucker rat plasma lipids are known to be elevated, although this abnormal lipid pattern does not lead to atherosclerosis. Accordingly, the hyperlipoproteinaemia occurring in obese Zucker rats appears not to affect the endothelium-dependent relaxation studied in our experiments with metacholine.

DISCUSSION As outlined in the Introduction the simultaneous occurrence of hypertension and diabetes is a relevant

103

problem that deserves intensive studies, including pharmacological investigations. The requirement of animal models for experimental biochemical, pathophysiological and pharmacological studies has been met, at least in part, by the introduction of the STZ-SHR and the obese Zucker rat. The STZ-SHR, developed a few years ago [7, 8], has been characterized in more detail by our group. The animals thus treated develop a hyperglycaemic syndrome, associated with other biochemical and morphological changes that to some extent approach IDDM (type 1 diabetes) combined with hypertension as occurring in humans. The model is easy to handle, reproducible and suitable for pharmacological and biochemical investigators. The rats with simultaneous diabetes and hypertension survived in sufficient numbers for experimental use during the incubation time 8 weeks prior to the experiments, as reflected by a low mortality rate. Control animals with either hypertension or diabetes alone are readily available and adequate for comparative purposes. The obese (Fa/?) Zucker rat, now readily available, is characterized by the simultaneous occurrence of obesity, hyperglycaemia, hyperinsulinaemia, hyperlipidaemia and moderate hypertension. As such it approaches the patient with NIDDM (type 2 diabetes) who is simultaneously hypertensive. Lean (fa/fa) Zucker rats are suitable and readily available control animals with respect to the obese animals. The obese Zucker rats of 22 weeks of age used in our experiments do not show relevant vascular damage, in spite of their hyperlipidaemia, moderate hypertension and hyperglycaemia. It may be that older animals which have been exposed to various noxious factors for longer periods of time will indeed develop relevant vascular damage. Pharmacological investigations have so far been focused predominantly on the STZ-SHR and its organs and tissues. Experiments with isolated mesenteric artery preparations would indicate endothelial dysfunction associated with the diabetic state. However, experiments with isolated small arteries (Mulvany technique) did not clearly indicate the existence of pharmacologically relevant, generalized endothelial dysfunction associated with diabetes. Experiments with isolated hearts taken from STZSHR indicate that the combination of hypertension and diabetes leads to progressive cardiac deterioration, which influences the pharmacodynamic characteristics of such hearts. Changes in calcium handling probably contribute to the aberrant pharmacodynamic pattern of STZ-SHR hearts. The cardiodepressant effect of nifedipine on isolated working heart preparations was hardly influenced by the hypertensive and/or diabetic state of the donor animals. Interestingly, nifedipine proved clearly protective against ischaemic damage in hearts from diabetic rats, but not in hearts from non-diabetic animals. The diabetic state

104

has been compared with the process of preconditioning. The pressor responses to various vasoconstrictor agents in pithed rat preparations made of STZ-SHR proved enhanced, probably as a result of the vascular hypertrophy associated with the hypertensive state. Interestingly, diabetes had an opposite, blunting effect on these amplified pressor responses, which so far cannot be explained in detail. It seems likely, however, that changes in cellular calcium handling contribute to this blunting influence of the diabetic state on pressor responses. A few data concerning the pharmacological profile of obese Zucker rats and their tissues are beginning to emerge. Vascular damage in these animals appears to develop very slowly, in spite of the presence of several vascular risk factors. In animals of 22 weeks of age we found no pharmacodynamic changes in the resistance vessels of obese Zucker rats, although changes in the aorta have been described.

CONCLUSIONS AND PERSPECTIVES Hypertension and diabetes can now be evoked separately or in combination in at least two types of animal models, which reasonably resemble and approach the two major categories of diabetes in humans (IDDM and NIDDM), combined with hypertension. Both models are easy to handle and reproducible, whereas adequate control animals are available for comparison. The STZ-SHR is now characterized in detail and its pharmacological analysis is well under way, although numerous problems remain to be solved, especially at the molecular–cellular level. The obese Zucker rat has been introduced more recently and very little of its pharmacological profile has been characterized. The resemblance of this model to the human NIDDM+hypertension syndrome indicates that the obese Zucker rat deserves more attention and also in pharmacological research.

REFERENCES 1. Yamori Y. Development of the spontaneously hypertensive rat (SHR) and of various spontaneous rat models, and their implications. In: de Jong W, ed. Handbook of hypertension, Vol. 4. Amsterdam: Elsevier, 1984: 224–39. 2. Yamori Y. The stroke-prone spontaneously hypertensive rat: contribution to risk factor analysis and prevention of hypertensive diseases. In: de Jong W, eds. Handbook of hypertension, Vol. 4. Amsterdam: Elsevier, 1984: 240–55. 3. Leenen FHH, Myers MG. Pressor mechanisms in renovascular rats. In: de Jong W, ed. Handbook of hypertension, Vol. 4. Amsterdam: Elsevier, 1984: 24–53. 4. Karasik A, Hattori M. Use of animal models in the study of diabetes. In: Kahn CR, Weir GC, eds. Joslin’s diabetes mellitus, 13th edn. Philadelphia, PA: Lea & Febiger, 1994: 217–350.

Pharmacological Research, Vol. 33, No. 2, 1996

5. Fischer LJ. Drugs and chemicals that produce diabetes. Trends Pharmacol Sci 1985; 6: 72–5. 6. Chappel CI, Chappel WR. The discovery and development of the BB rat colony: an animal model of spontaneous diabetes mellitus. Metabolism 1983; 32: 8–10. 7. Factor SM, Bhan RAJ, Minase T, Wolinsky H, Sonnenblick EH. Hypertensive–diabetic cardiomyopathy in the rat, an experimental model of human disease. Am J Pathol 1981; 102: 219–28. 8. Rodgers RL. Depressor effect of diabetes in the spontaneously hypertensive rat: associated changes in heart performance. Can J Physiol Pharmacol 1986; 64: 1177–84. 9. Clark JB, Palmer J, Shaw WN. The diabetic Zucker fatty rat (41611). Proc Soc Exp Biol Med 1983; 173: 68–75. 10. Kurtz TW, Morris RC, Pershadsingh HA. The Zucker fatty rat as a genetic model of obesity and hypertension. Hypertension 1989; 13: 897–901. 11. Bray GA. The Zucker-fatty rat: a review. Fed Proc 1977; 148–53. 12. Kasiske BL, O’Donnell MP, Keane WF. The Zucker rat model of obesity, insulin resistance, hyperlipidemia, and renal injury. Hypertension 1992; 19: I-110–5. 13. Hendriks MGC, Kam KL, Pijl AJ, Pfaffendorf M, van Zwieten PA. The effects of hypertension and diabetes mellitus on the vascular reactivity of resistance arteries. Blood Press 1993; 2: 69–76. 14. Kam KL, Hendriks MGC, Pijl AJ, Pfaffendorf M, van Zwieten PA. Spontaneously hypertensive rats with diabetes: effects of some vasoconstrictors on isolated small arteries. J Hypertens 1993; 11 (Suppl. 5): S120–1. 15. Kam KL, Hendriks MGC, Pijl AJ, van Marle J, van Veen HA, Pfaffendorf M, van Zwieten PA. Contractile responses to various stimuli in isolated resistance vessels from simultaneously hypertensive and streptozotocin-diabetic rats. J Cardiovasc Pharmacol 1996; 27: 167–75. 16. Pijl AJ, van der Wal AC, Mathy MJ, Kam KL, Hendriks MGC, Pfaffendorf M, van Zwieten PA. Streptozotocininduced diabetes mellitus in spontaneously hypertensive rats: a pathophysiological model for the combined effects of hypertension and diabetes. J Pharm Methods 1994; 32: 225–33. 17. Mulvany MJ, Hansen PK, Aalkjaer C. Direct evidence that the greater contractility of resistance vessels in spontaneously hypertensive rats is associated with a narrowed lumen, a thickened media, and an increased number of smooth muscle cell layers. Circ Res 1978; 43: 854–64. 18. Warshaw DM, Mulvany MJ, Halpern W. Mechanical and morphological properties of arterial resistance vessels in young and old spontaneously hypertensive rats. Circ Res 1979; 45: 250–9. 19. Mulvany MJ, Aalkjaer C, Christensen J. Changes in noradrenaline sensitivity and morphology of arterial resistance vessels during development of high blood pressure in spontaneously hypertensive rats. Hypertension 1980; 2: 664–71. 20. Regan TJ, Ettinger PO, Kahn MI, et al. Altered myocardial function and metabolism in chronic diabetes mellitus without ischemia in dogs. Circ Res 1974; 35: 222–37. 21. Fein FS, Sonnenblick EH. Diabetic cardiomyopathy. Prog Cardiovasc Dis 1985; 27: 255–70. 22. Factor SM, Minase T, Cho S, Fein F, Capasso JM, Sonnenblick EH. Coronary microvascular abnormalities in the hypertensive–diabetic rat, a primary cause of cardiomyopathy? Am J Pathol 1984; 116: 9–20. 23. Somani P, Singh HP, Saini RK, Rabinovitch A. Strepto-

Pharmacological Research, Vol. 33, No. 2, 1996

24.

25.

26. 27. 28. 29.

30. 31. 32. 33. 34. 35.

zotocin-induced diabetes in the spontaneously hypertensive rat. Metabolism 979; 28: 1075–7. Agraawal DK, McNeill JH. Effect of diabetes on vascular smooth muscle function in normotensive and spontaneously hypertensive rat mesenteric artery. Can J Physiol Pharmacol 1987; 65: 2274–80. Tomlinson KC, Gardiner SM, Hebden RA, Bennet T. Functional consequences of streptozotocin-induced diabetes mellitus, with particular reference to the cardiovascular system. Pharmacol Rev 1992; 44: 103–50. Zemel MB, Sowers JR, Shehin S, Walsh MF, Levy J. Impaired calcium metabolism associated with hypertension in Zucker obese rats. Metabolism 1990; 39: 704–8. McCaleb ML, Sredy J. Metabolic abnormalities of the hyperglycemic obese Zucker rat. Metabolism 1992; 41: 522–5. Paulson DJ, Tahiliani AG. Cardiovascular abnormalities associated with human and rodent obesity. Life Sci 1992; 51: 1557–69. Johnson IR, Curwen JO, Wilbraham JM, Hatton R. Differences in blood pressure, heart rate and vascular reactivity in the obese and lean Zucker rat. Br J Pharmacol 1994; 112 (Suppl.): 200P. Folkow B, Karlstrom G. Vascular reactivity in hypertension: importance of structural influences. J Cardiovasc Pharmacol 1987; 10 (Suppl. 4): 525–30. Folkow B. Structural factor in primary and secondary hypertension. Hypertension 1990; 16: 89–100. Jie K, van Brummelen P, van Zwieten PA. α -adrenoceptors in blood vessels of the human forearm. Prog Pharmacol 1986; 6: 37–45. van Zwieten PA. Adrenergic and muscarinic receptors. Classification, pathophysiological relevance and drug target. J Hypertens 1991; 9 (Suppl. 6): S18–27. Kam KL. Diabetes mellitus and/or hypertension in various pathological models (a pharmacological study). PhD Thesis. University of Amsterdam, 1994: 75–98. Kam KL, Pfaffendorf M, van Zwieten PA. Druginduced endothelium dependent and -independent relaxations in isolated resistance vessels taken from simultaneously hypertensive and streptozotocin-diabetic rats. Blood Press 1994; 3: 418–27.

105

36. Beenen OHM, Pfaffendorf M, van Zwieten PA. Contractile responses to various inotropic agents in isolated hearts obtained from hypertensive diabetic rats. Blood Press 1995; 4: 372–8. 37. Timmermans PBMWM, Mathy MJ, Wilffert B, Kalkman HO, Thoolen MJMC, de Jonge A, van Meel JCA, van Zwieten PA. Differential effect of calcium entry blockers on α 1-adrenoceptor mediated vasoconstriction in vivo. Naunyn-Schmiedeberg’s Arch Pharmacol 1983; 324: 239–45. 38. Pijl AJ, Hendriks MGC, Kam KL, Pfaffendorf M, van Zwieten PA. Anti-ischemic effects of nifedipine in isolated working heart preparations of healthy, diabetic and hypertensive rats. J Cardiovasc Pharmacol 1994; 23: 379–86. 39. Pijl AJ, Hendriks MGC, Kam KL, Pfaffendorf M, van Zwieten PA. Effects of R 56865 on postischemic ventricular function in isolated rat working heart preparations obtained from healthy, diabetic and hypertensive animals. Naunyn-Schmiedeberg’s Arch Pharmacol 1994; 349: 619–26. 40. Beenen OHM, Mathy MJ, Pfaffendorf M, van Zwieten PA. Influence of nifedipine on pressor responses induced by different α -adrenoceptor agonists and angiotensin II in pithed diabetic hypertensive rats. Submitted. 41. Kam KL, Pfaffendorf M, van Zwieten PA. Pharmacodynamic behaviour of isolated resistance vessels obtained from hypertensive diabetic rats. Fundam Clin Pharmacol 1996; in press. 42. Cox RH, Kikta DC. Age-related changes in thoracic aorta of obese Zucker rats. Am J Physiol 1992; 262: H1584–H1556. 43. Auget M, Delaflotte S, Braquet P. Increased influence of endothelium in obese Zucker rat aorta. J Pharm Pharmacol 1989; 41: 861–4. 44. Zemel MB, Peuler JD, Sowers JR, Simpson L. Hypertension in insulin-resistance Zucker obese rats is independent of sympathetic neural support. Am J Physiol 1992; 262: E368–73.