Decreased endothelial size and connexin expression in rat caudal arteries during hypertension.

OBJECTIVES: Hypertension is accompanied by endothelial dysfunction. The present study has investigated endothelial cell morphology and connexin expression in the caudal artery of the rat during the development of hypertension. METHODS: A significant increase in systolic blood pressure was detected from 9 weeks of age in spontaneously hypertensive male rats (SHR) compared to normotensive Wistar-Kyoto (WKY) rats, reaching a maximum by 11-12 weeks of age. Immunohistochemistry was used to quantify cell size and expression of connexins (Cxs) 37, 40 and 43 in the endothelium of prehypertensive (3-week-old) and hypertensive (12-week-old) rats. RESULTS: At 12 weeks, the size of endothelial cells and the expression of all three Cxs per endothelial cell were significantly less in SHR than WKY rats. At 3 weeks, there was no significant difference in cell size nor in the expression of Cxs 37 or 43; however, expression of Cx40 was significantly lower in SHR than in WKY rats. Between 3 and 12 weeks in WKY rats, there was no change in endothelial cell size, nor in the expression of Cxs 37, 40 and 43. In SHR, both cell size and Cx expression per endothelial cell were significantly decreased during the same developmental period, with a significant decrease in the density of Cx40 plaques. CONCLUSION: The development of hypertension in the SHR is accompanied by significant decreases in endothelial cell size and expression of Cx40, which may contribute to the endothelial dysfunction present in hypertension.

Contribution of central amiloride-sensitive transport systems to the development of hypertension in spontaneously hypertensive rats.

This study was conducted to examine if central amiloride-sensitive transport systems are involved in the development and/or maintenance of hypertension in spontaneously hypertensive rats (SHR). Either amiloride (75 microg/60 microl/day) or artificial cerebrospinal fluid (aCSF, 60 microl/day) was infused centrally (i.c.v.) for 4 weeks to development (4-5-weeks-old) and maintenance (10-12-weeks-old) phases of hypertension in SHR. In development phase, amiloride i.c.v. (n=14) blunted the elevation of blood pressure (BP) compared to aCSF i.c.v. (n=9) (amiloride vs. aCSF; after 3 weeks of i.c.v., 146+/-3 vs. 166+/-5 mmHg, P<0.001). The difference of BP at 3 weeks of i.c.v. was canceled after ganglionic block with hexamethonium (115+/-4 vs. 117+/-5 mmHg). Further, pressor responsiveness to norepinephrine was augmented in amiloride i.c.v. rats (amiloride, n=11 vs. aCSF, n=6; %Delta BP at 800 ng/kg/min.: 16.9+/-1.3 vs. 10.8+/-1.4 mmHg, P<0.05) and this augmentation disappeared after ganglionic block. Pressor responsiveness to angiotensin II and cumulative sodium balance did not differ in the two groups. Intravenous administration of amiloride at the same dose did not attenuate the development of hypertension. On the other hand, in maintenance phase, amiloride i.c.v. by the same protocol as in development phase had no effect on BP in SHR. Also, amiloride i.c.v. did not affect BP in normotensive Wistar-Kyoto rats. These results suggest that central amiloride-sensitive transport systems are involved in the development, but not in the maintenance, of hypertension in SHR through the modulation of autonomic neural mechanisms.

Myogenic tone in mesenteric arteries from spontaneously hypertensive rats.

To investigate myogenic tone during the developmental and established phases of hypertension, segments of distal (6th order) mesenteric arteries from spontaneously hypertensive rats (SHR) at 5 and 20 wk were isolated and pressurized in vitro and compared with vessels from age-matched Wistar-Kyoto (WKY) control animals. At 5 wk, tone was significantly enhanced in the SHR. At 20 wk tone was no longer significantly increased over a wide pressure range, although arteries from the SHR were able to maintain diameter at all pressures studied, whereas vessels from the WKY exhibited forced distension at 180 and 200 mmHg. From the relative slope of the pressure-diameter relationship (myogenic index), no increase in peak myogenic responsiveness was observed in arteries from the SHR at either time point. Passive lumen diameters were significantly decreased in arteries from SHR at both time points. From the total and passive midwall circumference-tension relationships, total tension was observed at a reduced midwall circumference in the SHR, but increased absolute levels of total tension were not observed. The normalized midwall circumference-tension relationships in the two strains revealed increased total tension due to active tension development at a reduced normalized circumference at 5 wk in the SHR. At 20 wk the normalized midwall circumference-tension relationships in the two strains were identical. These results demonstrate that myogenic tone in mesenteric arteries is enhanced during the development of hypertension but not when it is established, except at high intraluminal pressures.

Transforming growth factor-beta 1 in heart development.

Defined biochemical stimuli regulating neonatal ventricular myocyte (cardiomyocyte) development have not been established. Since cardiomyocytes stop proliferating during the first 3-5 days of age in the rodent, locally generated 'anti-proliferative' and/or differentiation signals can be hypothesized. The transforming growth factor-beta (TGF-beta) family of peptides are multifunctional regulators of proliferation and differentiation of many different cell types. We have determined in neonatal and maturing rat hearts that TGF-beta 1 gene expression occurs in pups of both normotensive (Wistar Kyoto, WKY) and hypertrophy-prone rats (spontaneously hypertensive, SHR). TGF-beta 1 transcript levels were readily apparent in total ventricular RNA from SHR pups within 1 day of age and elevated in 3-7 day old WKY and SHR hearts when cardiomyocyte proliferation indices are diminished. TGF-beta 1 transcript levels remain at a 'relatively' high level throughout maturation and into adulthood in both strains. Further, TGF-beta 1 transcripts were localized to cardiomyocytes of neonatal rat ventricular tissue sections by in situ hybridization. Immunoreactive TGF-beta was co-localized to the intracellular compartment of neonatal cardiomyocytes at the light and electron microscopic level. In vitro analysis using primary cultures of fetal and neonatal cardiomyocytes indicated that TGF-beta s inhibit mitogen stimulated DNA synthesis and thymidine incorporation. From these data, we propose that locally generated TGF-beta s may act as autocrine and/or paracrine regulators of cardiomyocyte proliferation and differentiation as intrinsic components of a multifaceted biochemical regulatory process governing heart development.

Ontogeny of absence-like and tonic seizures in the spontaneously epileptic rat.

The ontogeny of epileptic seizures in spontaneously epileptic rats (SER; zi/zi, tm/tm) was studied by examining behaviour and electroencephalogram (EEG) simultaneously. Weight gain and survival time were also studied. Compared with the control Kyo:Wistar rats, SER showed a much smaller increase in body weight. All male and female SER died before 20 and 18 weeks of age, respectively. Body tremor was observed at 2 weeks of age but disappeared after 11 weeks. Staggering gait appeared after 7 weeks of age, and intensified with age. Absence-like seizures characterized by paroxysmal appearance of 5-7 Hz spike-wave-like complexes were observed in the cortical or hippocampal EEG after 5 weeks of age, and tonic seizures with low voltage fast waves were observed after 6 weeks of age. All SER exhibited both absence-like and tonic seizures with high frequencies from 12 weeks of age. Differences with other spontaneous rat models of epilepsy and application methods for estimating seizure-inhibitory effects of anti-epileptic drugs are discussed.

Calcium uptake by duodenal enterocytes isolated from young and mature SHR and WKY rats: influence of dietary calcium.

Intestinal calcium (Ca2+) transport was examined at the cellular level using duodenal enterocytes isolated from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). Compartmental analysis of 45Ca2+ uptake was performed on enterocytes isolated from young (12- to 14-wk-old) and mature animals (24- to 26-wk-old) fed either normal (1%) or high (2%) calcium diets. Intracellular Ca2+ flux (Jc) was reduced in SHR compared with WKY for both young (0.67 +/- 0.05 vs. 1.08 +/- 0.08 nmol Ca2+.mg protein-1.min-1 P less than 0.01) and mature (0.39 +/- 0.03 vs. 0.71 +/- 0.05 nmol Ca2+.mg protein-1.min-1; P less than 0.001) animals on a normal calcium diet. On a high-calcium diet, this strain difference of Jc disappeared in the young rats (0.87 +/- 0.09 vs. 1.06 +/- 0.06 nmol Ca2+.mg protein-1.min-1, NS). In mature SHR, the high-calcium diet stimulated Jc, whereas it lowered it in mature WKY resulting in a similar flux for both strains (0.56 +/- 0.05 vs. 0.49 +/- 0.05 nmol Ca2+.mg protein-1.min-1, NS). Young SHR had a lower intracellular Ca2+ pool compared with WKY. This defect was corrected by a high-calcium diet. The membrane Ca2+ flux (Jm) was lower in mature SHR than WKY fed a normal calcium diet (P less than 0.02); Jm increased to the control value (P less than 0.05) in the SHR on a high-calcium diet. Diet-induced changes of plasma 1,25(OH)2 vitamin D levels in the SHR did not parallel the observed changes of intestinal Ca2+ fluxes. Thus duodenal enterocytes from SHR appear to have an intrinsic alteration of Ca2+ transport that can be corrected in part by a higher calcium diet.

Defective modulation of noradrenergic neurotransmission by endogenous prostaglandins in aging spontaneously hypertensive rats.

Sympathetic nerve stimulation causes a greater vascular response in spontaneously hypertensive rats (SHR) compared to Wistar-Kyoto normotensive rats (WKY), i.e., noradrenergic neurotransmission is enhanced in SHR. Prejunctional and/or postjunctional defects in the regulation of noradrenergic neurotransmission by endogenous prostaglandins could contribute to the increased responsiveness to sympathetic nerve stimulation in SHR. This hypothesis was tested by comparing the effects in SHR vs. WKY of inhibition of cyclooxygenase on vascular responses to periarterial nerve stimulation (PNS), norepinephrine (NE) and angiotensin II (ang II) in the in situ blood perfused rat mesentery. The cyclooxygenase inhibitor, indomethacin, potentiated vascular responses to PNS and NE similarly in 16-week old SHR vs. age-matched WKY. However, in this age group, indomethacin enhanced responses to ang II more in SHR compared with WKY. To determine whether chronic exposure of the vasculature to high blood pressure might alter the physiological significance of prostaglandin-mediated regulation of noradrenergic neurotransmission in vivo, additional studies were conducted in SHR and WKY that were 25 weeks old. In this age group, neither indomethacin nor ibuprofen, an alternative cyclooxygenase inhibitor, significantly potentiated responses to either PNS or NE in SHR, whereas in WKY both indomethacin and ibuprofen potentiated responses to PNS and NE. Also, in these older animals, indomethacin and ibuprofen enhanced responses to ang II equally in SHR vs. WKY. These findings indicate that in aging SHR prostaglandin-mediated regulation of vascular responses to sympathetic nerve stimulation becomes defective. This defect may contribute to the worsening of high blood pressure with age and may be involved in some of the vascular pathology associated with hypertension.

PGE2 synthesis in cultured renal papillary collecting tubule cells from young and aged spontaneously hypertensive rats.

To investigate whether altered renal medullary prostaglandin (PG) synthesis is involved in the development of hypertension in spontaneously hypertensive rats (SHR), we compared the capacity of PGE2 synthesis in cultured renal papillary collecting tubule cells from young (4-week-old) and aged (16-week-old) SHR and control Wistar-Kyoto rats (WKY). Basal levels of PGE2 synthesis were lower in young SHR cells than in WKY cells (p less than 0.001). Arachidonic acid-stimulated PGE2 synthesis, however, had a slight tendency to be higher in SHR cells than in WKY cells. Bradykinin- and A23187-stimulated PGE2 synthesis were similar in both strains. Basal levels of cyclic AMP were also lower in young SHR cells than in WKY cells (p less than 0.001), but the cAMP response to exogenous PGE2 was equal between the strains. In papillary collecting tubule cells from aged rats, basal levels of PGE2 and cyclic AMP as corrected for cellular protein were significantly lower than those in young rats, but there was no difference between the strains. Urinary excretion of PGE2 and thromboxane B2 was equal in aged SHR and WKY. These results suggest that papillary collecting tubule of young SHR and WKY may differ in the metabolism of PGE2 and cyclic AMP. This difference may be attributed to the possible defect in arachidonate availability in SHR.

Blood pressure, salt appetite and mortality of genetically hypertensive and normotensive rats maintained on high and low salt diets from weaning.

1. Blood pressure, bodyweight, saline preference and mortality rate were examined in spontaneously hypertensive rats (SHR) of the Okamoto strain and normotensive control Wistar-Kyoto (WKY) rats maintained on low (0.1% NaCl w/w), control (0.8% w/w) and high (3% w/w) salt diets from weaning until 6 months of age. 2. The growth rate of SHR on high salt diet was not significantly different from that on control diet but SHR maintained on a low salt diet exhibited a markedly reduced growth rate. While the growth rate of WKY on low salt diet was not significantly different from that on control diet, the bodyweights of WKY on high salt diet were significantly greater than those of animals on control diet. 3. While low salt diet markedly attenuated the development of hypertension in the SHR, high salt diet significantly exacerbated the blood pressure of this strain. Neither high nor low salt diet altered the blood pressure of WKY. 4. SHR on high and low salt diets had an increased mortality rate compared with SHR on control salt diet but these differences were of slight statistical significance. Conversely, WKY on all three diets exhibited similar mortalities over the 6-month observation period. There were no significant differences in mortalities between SHR and WKY on any diet. 5. The preference for 0.9% saline, when offered as a choice with water, was not significantly different between SHR on the different diets. WKY on high salt diet, however, exhibited a significantly reduced preference for saline over the 10-day test period compared with animals on control or low salt diet. 6. Thus dietary salt modulates the hypertension of SHR but not the blood pressure of WKY. SHR would appear to require more dietary sodium for normal growth and perhaps full expression of its hypertension. The higher and lower blood pressures of the SHR on high and low salt diet, respectively, were associated with increased mortality, which was a trend not seen in the WKY.

Neurotransmitters and neuropeptides in blood pressure regulation in the spontaneously hypertensive rat.

Studies of the roles played by neurotransmitters in the development of hypertension in the spontaneously hypertensive (SHR) rat are complicated by the presence of genetic differences between SHR and normotensive control rats, which are not related to differences in blood pressure. One approach that may be used in an attempt to overcome this difficulty is to study the manner in which neurotransmitter and metabolite levels change with age, and to relate these changes to alterations in blood pressure with ageing. Noradrenaline (NA) levels in the brainstem and spinal cord of SHR and Wistar Kyoto rats fell with age, while 3,4-dihydroxyphenylethyleneglycol (DHPG) levels (a neuronal metabolite of noradrenaline) remained constant. Similar changes were seen when NA and DHPG levels were measured in the discrete brainstem A1, A2, and C2 region, and when adrenaline, NA, and DHPG levels were examined in the C1 region. Differences in age-related changes of neuropeptide Y (NPY) levels were also found in the ventromedial nucleus of the hypothalamus and the locus coeruleus, and of beta-endorphin in the anterior hypothalamic nucleus, the paragigantocellular nucleus of the brainstem, and the locus coeruleus. These changes may indicate either a progressive increase in the activity of neurons in the sympathoexcitatory C1 region or a progressive reduction in the activity of vasodepressor A1, A2, and C2 regions with ageing, or both. However, changes in catecholamines and metabolites with age were similar in both strains and therefore cannot readily explain the more rapid rise in blood pressure with ageing in SHR rats.

Biological variability in Wistar-Kyoto rats. Implications for research with the spontaneously hypertensive rat.

The spontaneously hypertensive rat (SHR) initially bred in Kyoto is the most widely studied animal model of essential hypertension. As controls for the SHR, most workers have used normotensive descendants of Wistar rats from the colony in Kyoto from which the SHR strain was derived (Wistar-Kyoto rats, WKY). But the presumption that WKY are serviceable controls for SHR rests on the tacit assumption that all WKY constitute a single inbred strain. It appears, however, that whereas the National Institutes of Health distributed breeding stocks of SHR after they had been fully inbred (i.e., after 20 generations of brother-sister mating), the breeding stocks of WKY were distributed before they had been fully inbred. Accordingly, the biological variability of WKY may be greater than that of SHR. To investigate this possibility, we obtained SHR and WKY from two of the largest commercial suppliers in the United States and systematically measured the growth rate and blood pressure of these rats under identical physical and metabolic conditions. We found that WKY from one source differed from those of the other in both growth rate and blood pressure. In contrast, the SHR from the two suppliers were not different with respect to either growth rate or blood pressure. Because the National Institutes of Health may have distributed breeding stocks of WKY as early as the F6 generation, it is possible that rats currently designated as WKY do not constitute a single inbred strain. Thus, interpretation of studies employing "the Wistar-Kyoto rat strain" as a control for the SHR may be much more problematic than has previously been recognized.

Body weight of the spontaneously hypertensive rat during the suckling and weanling periods.

The body weight of the spontaneously hypertensive rat (SHR) during the suckling, birth to 3 weeks of age, and weanling period, through 6 weeks of age, is compared to that of its normotensive controls, the Wistar Kyoto (WKY) and Wistar (WR) rats. Litters were normalized to 6 pups per dam just after birth. The WR is larger than the SHR and WKY at all time points examined. The WKY is similar in weight to the SHR at birth but larger than the SHR at the other time points studied. These findings suggest the WR is inherently a larger rat than the SHR and WKY during the suckling and weanling periods while the development of a weight difference between the SHR and WKY after birth suggest an extrauterine influence.

Characteristics of spontaneously hypertensive, Wistar Kyoto and Munich Wistar rats bred in Brazil.

The breeding of imported strains of isogenic rats was started in 1981 because of the lack of experimental rat models in Brazil. The imported strains were: Spontaneously Hypertensive Rats (SHR) and their normotensive controls Wistar Kyoto Rats (WKY) from the National Institutes of Health, Bethesda, MD, and Munich Wistar (MW) rats which present superficial renal glomeruli, from Simonsen Laboratories, Gilroy, CA. Breeding of these strains was carried out without strict barriers (conventional breeding), under Standard Operating Procedures and strict inbreeding. Environmental factors such as ration, light, temperature, type of shavings and bedding, size of cages and their population were constant. Body weight growth curves were constructed for the three strains. The productivity of imported rats and local breeding colonies in 1981, 1982 and 1983 was compared on the basis of the following parameters: mean litter size, productivity of the females, pre- and post-weaning mortality, and effective yield. Systolic blood pressure was also measured for SHR and WKY rats. MW rats showed a high and relatively stable reproduction performance. The productivity of SHR and especially WKY animals declined progressively during the first three years, making the breeding of these strains of isogenic rats very difficult.