Adult Wistar-Kyoto Rats Research Articles

Similar genetic mechanisms may underlie sleep-wake states in neonatal and adult rats.

Genetic differences in the characteristics of sleep-wake states in adult animals offer a potential window for examining how the neonatal and adult behavioural states are related to one another. Our recent finding that adult Wistar-Kyoto (WKY) rats show pronounced genetic differences in sleep-wake patterns relative to the Wistar (WIS) control strain led us to investigate the relationship between these behavioural states in neonates and adults in a longitudinal study in these two strains of rats. Similar pronounced differences in the sleep-wake states were observed between WKY and WIS rats in neonatal and in adult animals. At both ages, WKY rats spent more time in activesleep (AS) and rapid eye movement sleep (REMS) and less time in quiet sleep (QS) and non-REM sleep (NREMS) than WIS rats, and the sleep-wake states were more fragmented in neonatal and adult WKY rats. While it is not known how neonatal AS and QS are physiologically related to adult REMS and NREMS, respectively, the finding of similar differences in the amounts of sleep-wake states in neonatal and adult WKY and WIS rats argues strongly that at some level they are controlled by similar genetic as well as cellular/physiological mechanisms.

Relevance of endothelium-derived hyperpolarizing factor in the effects of hypertension on rat coronary relaxations.

To evaluate the relative participation of nitric oxide (NO), endothelium-derived hyperpolarizing factor (EDHF) and prostanoids in the relaxing responses induced by acetylcholine and isoproterenol in isolated coronary arteries from adult Wistar- Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). Male adult WKY rats and SHR were used in the study. Segments from left coronary arteries (approximately 350-380 microm internal diameter and 2 mm long) were mounted in an isometric myograph and pre-contracted with serotonin. Dose-response curves to acetylcholine and isoproterenol were carried out in absence and presence of the NO synthesis inhibitor NG-nitro-L-arginine methyl ester (LNAME), the inhibitor of the cyclo-oxygenase, indomethacin and KCI. Areas under the respective dose-response curves were used to calculate the approximate relative participation of NO, EDHF and prostanoids. Relaxations to either acetylcholine or isoproterenol were lower in SHR than in WKY rats. In WKY rats, presence of LNAME diminished (P< 0.05) relaxation to acetylcholine from 10(-9) to 10(-6) mol/l, and induced a contracting response at 10(-5) and 10(-4) mol/l of acetylcholine. Addition of indomethacin did not significantly affect dose-related relaxation to acetylcholine 10(-9) to 10(-6) mol/l in WKY rats, and reduced (P < 0.05) the contracting response observed at 10(-5) mol/l of acetylcholine. In SHR, addition of LNAME markedly reduced (P< 0.05) acetylcholine relaxations, but did not produce any contracting effect. Addition of indomethacin on top of LNAME slightly (P< 0.05) enhanced relaxing response to acetylcholine in SHR. Presence of LNAME in the media diminished (P < 0.05) relaxation to isoproterenol in both WKY rats and SHR. Addition of indomethacin on top of LNAME increased (P< 0.05) isoproterenol-relaxing response to levels similar to and higher than control conditions in WKY rats and SHR, respectively. Addition of KCI blunted both acetylcholine- and isoproterenol-relaxations in both groups. NO and EDHF are the main endothelium-derived relaxing factors underlying acetylcholine and isoproterenol relaxations in rat coronary arteries, respectively. EDHF reduction, and not only NO reduction play a key role in the diminished coronary relaxations induced by acetylcholine and isoproterenol in SHR. An arachidonic acid derivative with contracting activity released by acetylcholine and isoproterenol in a differential manner, could oppose the relaxing actions of NO and EDHF.

Resistance artery mechanics, structure, and extracellular components in spontaneously hypertensive rats : effects of angiotensin receptor antagonism and converting enzyme inhibition.

Altered vascular mechanics resulting from changes in collagen and integrins may influence resistance artery structure and function and, therefore, peripheral resistance and blood pressure in spontaneously hypertensive rats (SHR). Effects of age, angiotensin-converting enzyme inhibition (fosinopril, 10 to 30 mg/kg per day), and AT(1)-receptor antagonism (irbesartan, 50 mg/kg per day) on vascular structure, mechanics, and composition were assessed in SHR. Systolic blood pressure was elevated in young SHR (130+/-2 mm Hg) compared with Wistar-Kyoto (WKY) rats (106+/-2 mm Hg). In adult SHR, the rise in systolic blood pressure (44+/-3 mm Hg) was blunted by fosinopril (18+/-1 mm Hg) and irbesartan (9+/-3 mm Hg). Lumen diameter of mesenteric resistance arteries was smaller and media/lumen ratio was greater in young and adult SHR versus WKY rats. Growth index was 24% in untreated adult SHR versus WKY rats; these values were -35% for fosinopril-treated and -29% for irbesartan-treated SHR versus untreated SHR. Isobaric wall stiffness was normal despite increased stiffness of wall components in adult SHR vessels. Irbesartan partially prevented stiffening of wall components in SHR. The collagen/elastin ratio was greater in adult SHR vessels (6.5+/-1.3) than in WKY (3.2+/-0.4) vessels. Expression of alpha(v)beta(3) and alpha(5)beta(1) integrins was increased in SHR aged 20 versus 6 weeks. Expression of alpha(5)beta(1) integrins was lower in young SHR, and alpha(v)beta(3) integrins were overexpressed in adult SHR versus WKY rats. Irbesartan and fosinopril attenuated differences in the collagen/elastin ratio and integrin expression. Wall components of mesenteric resistance arteries stiffen with age in SHR. Interrupting the renin-angiotensin system has normalizing effects on integrin expression and composition, stiffness, and growth of the arterial wall.

Visualisation of AMPA binding sites in the brain stem of normotensive and hypertensive rats

The present study has employed in vitro receptor autoradiography with ( S)-[ 3 H ]-5-fluorowillardiine (10 nM) to visualise the presence of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) binding sites in the brain stems of adult (16–18 weeks) normotensive (Wistar–Kyoto (WKY) and Don Ryu (DRY)) and Spontaneously Hypertensive (SHR) rats. Similar topographic distribution and density of ( S)-[ 3 H ]-5-fluorowillardiine binding was observed in the nucleus tractus solitarius (NTS) of all three strains. Specific ( S)-[ 3 H ]-5-fluorowillardiine binding sites were also visualised in sections of nodose ganglion from adult WKY rats, demonstrating that vagal afferent perikarya possess AMPA binding sites. However, while unilateral vagal deafferentation did not result in a significant decrease in binding site density in the caudal half of the rat NTS, the visualisation of AMPA binding sites on the nodose ganglion is consistent with the existence of a population of binding sites on vagal afferent terminals. In the caudal half of the rat NTS, AMPA binding sites appear to be predominantly postsynaptic in nature.

Patterns of Adrenoceptor Change During Liver Regeneration of the Wistar Kyoto Rat: A Binding Study

Background Adrenoceptors have been involved in the regulation of hepatocyte proliferation after partial hepatectomy, as well as in primary culture. This report characterizes α 1- and β-adrenoceptor change during the time-course of liver regeneration in adult Wistar Kyoto rats. Methods Saturation binding assays with [ 3H]prazosin or [ 3H]dihydroalprenolol (for α 1- and β-adrenoceptors, respectively) were done in liver plasma membranes from 6-month-old rats subjected to 70% hepatectomy followed by hepatic regeneration. Results [ 3H]Prazosin and [ 3H]dihydroalprenolol binding gave control B max values of 101 ± 10 and 12 ± 1 fmol/mg protein and K d of 0.50 ± 0.10 and 4.1 ± 0.4 nM for α 1- and β-adrenoceptors, respectively. α 1-Adrenoceptor number and K d increased at 24 and 48 h and returned to control values at 72 and 96 h after surgery, whereas β-adrenoceptors augmented at 48 and 72 h, with a K d change at 24 and 48 h posthepatectomy. Conclusions These results suggest that dual control of α 1- and β-adrenoceptor membrane expression could be involved in different steps during hepatocyte proliferation, and that Wistar Kyoto rats have a different adrenoceptor pattern expression from other rat strains.

Increased expression of Ca2+-sensitive K+ channels in aorta of hypertensive rats.

Potassium efflux through Ca2+-sensitive K+ channels (K[Ca] channels) is increased in arterial smooth muscle cells from hypertensive rats, but the molecular mechanism is unknown. The goal of this study was to compare the levels of K(Ca) channel current between aortic smooth muscle cells from adult Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) and then use Western blot methods and ribonuclease protection assays to examine the expression and mRNA levels for the K(Ca) channel in these same vascular tissues. Whole-cell patch-clamp methods indicated a larger component of K(Ca) channel current, sensitive to block by iberiotoxin (100 nmol/L), in single aortic smooth muscle cells from SHR compared with WKY. Subsequent Western blot analysis using a site-specific antibody (anti-alpha[913-926]) directed against the S9/S10 linker of the alpha-subunit of the K(Ca), channel revealed a 125-kD immunoreactive band in lanes loaded with either WKY or SHR aortic muscle membranes. The immunoreactive density of this band, which corresponded to the known molecular size of the alpha-subunit, was 2.2-fold greater in lanes loaded with aortic smooth muscle membranes from the hypertensive animals. However, despite this evidence for an increased expression and functional enhancement of K(Ca) channels in aortic smooth muscle membranes of SHR, ribonuclease protection assays with a 32P-labeled riboprobe targeted against the S9/S10 linker of the K(Ca) channel alpha-subunit revealed no difference in mRNA levels for the alpha-subunit between WKY and SHR aortic tissue. These findings provide initial evidence that (1) an increased expression of K(Ca) channels may be a mechanism for the enhanced K(Ca) current in aortic smooth muscle membranes of SHR, and (2) the upregulation of K(Ca) channels in arterial muscle membranes during hypertension, which is regarded as a homeostatic mechanism for buffering vascular excitability, may rely on posttranscriptional events.

Validation of a continuous baroreceptor reflex sensitivity index calculated from spontaneous fluctuations of blood pressure and pulse interval in rats.

To develop and validate a technique for the continuous computerized calculation of the baroreceptor reflex sensitivity (BRS) of the heart rate in rats. The BRS was calculated from spontaneous changes in blood pressure and pulse interval using spectral analysis as well as time-series techniques. The BRS values obtained with these techniques were compared with those obtained by standard pharmacological methods. The blood pressure and pulse interval in adult Wistar-Kyoto (WKY) rats were recorded on a beat-to-beat basis for two consecutive 30 min periods. During one of these periods the BRS was determined pharmacologically by injections of nitroprusside and phenylephrine. Measurements were performed after administration of saline as vehicle or during manipulation of the autonomic nervous system by infusion of metoprolol, methyl-atropine and hexamethonium. Sequential time-series methods for continuous BRS calculation were tested for 24 h periods in intact WKY rats as well as in WKY rats that had been subjected to sino-aortic denervation or to electrical lesioning of the nucleus tractus solitarius. The correlation coefficient between BRS values in intact WKY rats derived from the pharmacological method and those from spectral analysis techniques was low (R2 = 0.16). The correlation coefficient between BRS values from the pharmacological method and those from the developed time-series method was higher (R2 = 0.64). The BRS measured using the latter method was found to vary over 24 h with the highest values during the sleeping period. After surgical elimination of the baroreflex, the algorithm returned BRS values close to zero throughout the 24 h period. The BRS estimate was found to be a measure of the parasympathetic rather than of the sympathetic component of the baroreceptor reflex. The developed time-series method calculates an index of the gain of the cardiac baroreflex in rats faithfully. This method can be implemented in data acquisition software, allowing continuous on-line monitoring of the cardiac baroreflex gain.

Increased potency of neuropeptide y to antagonize α2-adrenoceptor function in the nucleus tractus solitarii of the spontaneously hypertensive rat

The regulation by neuropeptide Y of α2-adrenoceptors in the nucleus tractus solitarii was evaluated in the adult normotensive Wistar Kyoto rat and the adult spontaneously hypertensive rat. The microinjection of a submaximal dose of l-noradrenaline (800pmol in 50nl) alone into the nucleus tractus solitarii produced a significant reduction in the mean arterial blood pressure in either strain. The threshold dose (1pmol in 50nl) of neuropeptide Y(1–36) for the vasodepressor response in the Wistar Kyoto rat was five times higher than that (0.2pmol in 50nl) in the spontaneously hypertensive rat. Furthermore, neuropeptide Y(1–36) at 0.2pmol in 50nl could significantly counteract the vasodepressor response to l-noradrenaline (800pmol in 50nl) in the spontaneously hypertensive rat, but not in the Wistar Kyoto rat, in which 1pmol in 50nl of neuropeptide Y(1–36) must be employed to counteract the vasodepressor response to l-noradrenaline (800pmol in 50nl), although the vasodepressor responses are of a similar magnitude. The in situ hybridization and quantitative receptor autoradiographical experiments showed that the α2A-adrenoceptor messenger RNA levels and the Bmax value of the α2-adrenoceptor agonist [3H]p-aminoclonidine binding sites measured in the nucleus tractus solitarii of the spontaneously hypertensive rat were substantially lower than those in the Wistar Kyoto rat. The quantitative receptor autoradiographical results were consistent with the cardiovascular results and showed that in the spontaneously hypertensive rat, neuropeptide Y(1–36) at 1nM led to a significant increase in the Kd value of [3H]p-aminoclonidine binding sites. In the Wistar Kyoto rat, neuropeptide Y(1–36) produced this effect only at 10nM. The present study provides evidence for an increase of the potency of neuropeptide Y(1–36) to antagonistically modulate α2-adrenoceptors in the nucleus tractus solitarii of the spontaneously hypertensive rat. This enhanced antagonistic action may partly be related to a reduction in the number of α2A-adrenoceptors in the nucleus tractus solitarii of the spontaneously hypertensive rat, since a decrease has been observed in the α2A-adrenoceptor messenger RNA levels and the α2-adrenoceptor binding sites in the spontaneously hypertensive rat.This increased potency of neuropeptide Y(1–36) to antagonize α2-adrenoceptor function in the nucleus tractus solitarii of the spontaneously hypertensive rat may contribute to the development of high blood pressure in this hypertensive strain.

Decreased developmental cell death in sympathetic and spinal sensory nervous systems of the Kyoto spontaneously hypertensive rat.

To investigate the hypotheses that Kyoto spontaneously hypertensive rats (SHR) possess more sympathetic neurons than do normotensive Wistar-Kyoto (WKY) animals due to reduced perinatal cell death and that this is due to increased availability of the sympathetic survival neurotrophin, nerve growth factor. Total cell counts of neuron numbers were performed in neonatal and adult SHR and WKY rat superior cervical ganglia and correlated with counts of apoptotic cells. The values for sympathetic neuron numbers were compared with those for a spinal sensory ganglion. Immunocytochemistry was used to obtain more information about the phenotypes of neurons counted. Adult SHR sympathetic ganglia contained about 25% more sympathetic neurons than did those of WKY animals. Similar elevation of numbers was found both for neurons containing and for those devoid of neuropeptide Y. In neonatal animals, in contrast, there was no strain difference in sympathetic cell numbers but the number of apoptotic cells was reduced in SHR. Spinal sensory neuron numbers in adult SHR were elevated to a similar extent as were sympathetic neurons, but biochemical and morphometric data suggested that this change does not involve cells that are sensitive to nerve growth factor. Although our results support the view that there is reduced developmental cell death both in sympathetic and in sensory systems, they also suggest that this is unlikely to be due to a simple excess of nerve growth factor during development.

Angiotensin II regulation of tyrosine hydroxylase gene expression in the neuronal cultures of normotensive and spontaneously hypertensive rats.

In the present study we investigated the regulation of tyrosine hydroxylase (TH) by angiotensin II (Ang II) in an attempt to provide cellular and molecular evidence that this hormone has increased neuromodulatory actions in the spontaneously hypertensive (SH) rat brain. Neuronal cells in primary culture from the hypothalamus-brain stem of both normotensive [Wistar-Kyoto (WKY)] and SH rats have been used. These cultures mimic in vivo situations. Ang II caused a time-dependent increase in TH activity in WKY rat brain neurons. A maximal increase of 2.5-fold was observed with 100 nM Ang II in an actinomycin- and cycloheximide-dependent process. In addition, Ang II caused a parallel increase in TH messenger RNA (mRNA) levels, with a maximal stimulation of 5-fold in 4 h by 100 nM Ang II in WKY rat brain neurons. The stimulation of TH mRNA was mediated by the AT1 receptor subtype, resulted from an increase in its transcription, and involved activation of phospholipase C and protein kinase C. Antisense oligonucleotide for c-fos attenuated Ang II stimulation of TH mRNA in a time- and dose-dependent fashion, indicating an involvement of c-fos as a putative third messenger in Ang II stimulation of TH. Ang II also caused stimulation of TH activity and its mRNA levels in neuronal cultures of SH rat brain by a mechanism similar to that observed for neuronal cultures of WKY rat brain, involving AT1 receptors, protein kinase C, and c-fos. However, the stimulation of TH activity and that of TH mRNA were approximately 30% and 80% higher, respectively, in the SH rat brain neurons than those in the WKY rat brain neurons. In vivo experiments have been carried out to validate the elevated response of TH gene expression to Ang II in SH rat brain neuronal cultures. Ang II stimulated both TH activity and TH mRNA levels in the hypothalami and brain stems of adult WKY and SH rats. The level of stimulation in the brain of the SH rat was significantly higher than that in the WKY rat. These observations are consistent with an increase in AT1, receptor gene expression and suggest that increased TH gene expression could be the cellular/molecular basis for the greater neuromodulatory action of Ang II in the SH rat brain.

Comparison of 11β-hydroxysteroid dehydrogenase in spontaneously hypertensive and Wistar-Kyoto rats

11β-Hydroxysteroid dehydrogenase (11β-HSD) modulates glucocorticoid interactions with mineralocorticoid and glucocorticoid receptors in vivo, by converting 11β-hydroxyglucocorticoids to their inactive 11-ketone derivatives. Defective 11β-oxidation of glucocorticoids has been associated with hypertension. The objective of this study was to investigate whether 11β-HSD contributes to the occurrence of hypertension in spontaneously hypertensive rats (SHRs). The liver and kidney microsomal oxidations of corticosterone (the physiological glucocorticoid in rats) in organs from juvenile (3 weeks old) and adult (3 months old) SHR and Wistar-Kyoto (WKY) rats, with NAD and NADP, show no differences between rat strains. For cortisol, with NADP, adult SHRs show (1.3–3 times; P < 0.05) lower kidney microsomal oxidation rates. The liver microsomal reduction of cortisone shows remarkable interstrain differences: with NADH, reduction is conducted only by adult WKY rats, whereas with NADPH, juvenile animals show similar reduction rates, but at adulthood, only WKYs reduce cortisone. Using Western blot analysis with antibodies against 11β-HSD1, positive signals are obtained only for liver microsomes, appearing somewhat lower in SHRs for juvenile but not adult animals. Urinary corticosterone/11-dehydrocorticosterone ratios (measured in adult animals) are not different between rat strains, but are elevated after administration of corticosterone in both strains (although significant only in SHRs). The data provide no indications for exaggerated stimulation of renal corticosteroid receptors, due to modified 11β-HSD, in SHRs. However, the experiments suggest the existence of multiple 11β-HSDs, in addition to 11β-HSD1 and 11β-HSD2, some of which may be modified in SHR, but the nature and physiological role of these 11β-HSDs is unclear.

Increased activity of constitutive nitric oxide synthase in cardiac endothelium in spontaneous hypertension.

We analyzed the activity of nitric oxide synthase in the rat heart to study whether this activity is affected by high blood pressure. Hearts from young (3 to 4 weeks old) and adult (15 to 25 weeks old) Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) were excised and frozen in liquid nitrogen. The activities of Ca(2+)-dependent (cNOS) and Ca(2+)-independent (iNOS) were determined in homogenized tissues by measuring the conversion of [14C]-L-arginine to [14C]-L-citrulline in the presence or absence of either EGTA (1 mmol/L) or EGTA plus NG-nitro-L-arginine methyl ester (L-NAME, 1 mmol/L each). Arterial pressure was higher in adult SHR than in young SHR and WKY rats of both ages (P < .01). The cNOS activity was two to three times higher in hypertensive than in normotensive hearts (P = .01 to P = .04). No significant activity of iNOS was detected in any tissue. Studies of the right and left ventricles demonstrated a higher cNOS activity in the left sides of the hearts of adult SHR (P < .05). No differences were found in hearts from WKY rats. Selective removal of endocardial or coronary endothelial cells in hearts of SHR and WKY rats substantially reduced cNOS activity (P < .01). The cNOS activity is upregulated in cardiac endothelial cells of genetically hypertensive rats. The high activity of cardiac cNOS is related to increased arterial pressure of these animals. We propose that in the heart, endothelial cells respond with a higher production of nitric oxide as a compensatory mechanism against high blood pressure and its damaging effects in this organ.

In vitro production of angiotensin II by isolated glomeruli.

The glomerulus has several components of the renin-angiotensin system (RAS). The purpose of this study was to evaluate the ability of glomeruli isolated from adult Wistar-Kyoto rats to produce angiotensin II (ANG II). When isolated glomeruli were incubated in Krebs buffer, the peak concentration of immunoreactive angiotensin (ANG) in the incubation medium, representing simultaneous production and degradation, occurred after 15 min of incubation (3.98 +/- 0.34 pg.mg protein-1.15 min-1, of which 18% was ANG II. When 125I-labeled ANG II was incubated with isolated glomeruli, the half-life of ANG II was 6.06 min. Hence, we estimated ANG II production at 3.77 +/- 0.21 pg.mg protein-1.15 min-1. When angiotensinogen-rich serum was added to the incubation medium, ANG concentration at 15 min increased by 500-fold (1,978 +/- 44 pg.mg protein-1.15 min-1, P < 0.001). ANG concentration in the glomerular incubate responded to perturbations known to alter systemic RAS. Enalaprilat, chymostatin, propranolol, and renin antiserum decreased ANG concentration in glomerular incubate, whereas salt depletion increased this (P < 0.05). We conclude that the rat glomerulus can generate ANG II independent of neural, hormonal, or vascular control.

Kidney angiotensin II receptors and converting enzyme in neonatal and adult Wistar-Kyoto and spontaneously hypertensive rats

The aim of the present study was to correlate the development of the renin angiotensin system (RAS) in the kidney of the rat with the development of genetic hypertension. Immature (1-week-old) and adult (12-week-old) normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive kidney rats (SHR) were used for quantification of angiotensin II (ANG II) receptors and angiotensin converting enzyme (ACE) binding sites using quantitative autoradiography. In both neonatal and adult animals of either strain, ANG II receptors were of AT1 subtype. In all kidney areas of 1-week-old rats. ANG II receptor density was higher in SHR than WKY. Binding density increased with age in WKY rats; thus, in the glomeruli and the outer stripe of the outer medulla of 12-week-old WKY, binding was significantly higher than that present in age-matched SHR. [125I]351A binding to ACE was highest in the outer medulla and not detectable in glomeruli. In 1-week-old rats, binding to ACE was higher in WKY than in SHR strain. No differences in ACE binding were found between adult SHR and WKY rats, with the exception of the inner stripe of the outer medulla, where no binding was detected in SHR. Our results support the hypothesis that the RAS in kidney is developmentally regulated and is involved in the development and maintenance of genetic hypertension in SHR.

An improved procedure for the immunohistochemical localization of nerve growth factor-like immunoreactivity

Nerve growth factor (NGF) is a survival factor required by a number of neuronal populations including most post-ganglionic sympathetic neurones. NGF has been detected and quantified in many tissues but there is little information regarding its cellular localization. Although it has been argued that histological detection has proven difficult due to the low levels of NGF present, other factors may contribute to prevent its identification. In the present study, we report a method for the histological detection of NGF-like immunoreactivity in the rat superior cervical ganglia (SCG). Adult Wistar-Kyoto rats were perfused briefly with either a high or low pH buffer prior to fixation and routine immunohistochemistry. Polyclonal antibodies to native mouse NGF used in the present study recognized mouse NGF but not recombinant human neurotrophin 3 (rhNT3) or brain-derived neurotrophic factor (rhBDNF) by immunoblot analysis. NGF-like immunoreactivity was localized to most sympathetic neurones. Immunoreactivity was detected in the cytoplasm with dense labelling around nuclei. No stain was seen in sections incubated with normal sheep IgG or from animals perfused with phosphate buffer (pH 7.4) prior to fixation. In addition, axotomy resulted in the disappearance of NGF immunoreactivity which was confirmed by biochemical quantification. Finally, no NGF immunoreactivity was found in neurones of rats treated systemically with NGF antiserum 3 days earlier. Possible mechanisms underlying the improvement of NGF immunohistochemistry by pH manipulation before fixation are discussed.

A photoactivable source of relaxing factor in genetic hypertension.

Deendothelialized rings of rabbit aorta relax after exposure to UV light because of release of a relaxing factor that is similar if not identical to nitric oxide. We tested the hypothesis that production of the photo-induced relaxing factor is impaired in a rat model of genetic hypertension. Thoracic aortas were removed from adult Wistar-Kyoto rats and stroke-prone spontaneously hypertensive rats. The vessels were cut into rings, denuded of endothelium, and placed in a muscle bath for isometric force measurement. Rings were contracted with phenylephrine, and relaxation was measured after exposure to UV light. Aortic rings from stroke-prone spontaneously hypertensive rats relaxed to a greater extent after exposure to UV light than did rings from Wistar-Kyoto rats. An inhibitor of nitric oxide synthase (N omega-nitro-L-arginine) greatly potentiated the relaxation responses to light in both strains, and these enhanced relaxations were attenuated by tetraethylammonium chloride, potassium chloride, ouabain, or inhibitors of guanylate cyclase. These results suggest that UV irradiation induces relaxation in aortic smooth muscle that is greater in hypertensive than normotensive rats and is greatly enhanced after addition of inhibitors of nitric oxide production. Thus, the unidentified photo-induced relaxing factor is not solely nitric oxide but may also represent either a hyperpolarizing factor, because depolarization blocks the responses entirely, or possibly smooth muscle guanylate cyclase that might itself be photoactivable.

Glomerular Losartan (DuP 753)-Sensitive Angiotensin II Receptor Density Is Increased in Young Spontaneously Hypertensive Rats

There is increasing evidence that an activated intrarenal renin-angiotensin system (RAS) alters renal hemodynamics and fluid balance and that such events may lead to the development of hypertension. To examine the role of the glomerular RAS in the development of hypertension in the spontaneously hypertensive (SHR) rat, we studied angiotensin (ANG) II receptors in isolated glomeruli from young (4- to 5-wk-old) and adult (10- to 12-wk-old) SHR and from age-matched, normotensive Wistar-Kyoto (WKY) rats. Glomerular ANG II receptor density in young SHR is 3-fold higher than in age-matched WKY rats (2033 +/- 154 versus 742 +/- 151 receptors/microns2; p < 0.05) and 1.5-fold higher than in adult SHR and WKY rats (1128 +/- 85 and 1198 +/- 181 receptors/microns2, respectively; p < 0.05). Additional studies demonstrated that the differences in receptor density are not related to disparity in receptor occupancy and that they are also independent of systemic ANG levels. Suppression of RAS by ANG converting enzyme inhibitors resulted in a 3-fold increase in receptor density in young SHR rats and a 4.5-fold increase in young WKY rats; receptor density remained greater in young SHR rats (5915 +/- 318 versus 3358 +/- 234 receptors/microns2, p < 0.05). Furthermore, competitive binding experiments using the nonpeptide ANG II antagonists losartan (AT1) and PD 123319 (AT2) indicate that the greater ANG II receptor density in the young SHR rats represents an increase in the number of a single population of AT1 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of ramipril on contractile oscillations in arteries from genetically hypertensive rats.

We have tested the hypothesis that altered vascular reactivity, specifically the appearance of spontaneous and BayK 8644 (L-type voltage gated calcium channel agonist)-induced oscillations in the carotid artery and the sarcoplasmic reticulum Ca(2+)-ATPase inhibitor cyclopiazonic acid (CPA)-induced oscillations in the aorta from stroke-prone spontaneously hypertensive rats (SHRS), are dependent upon angiotensin II production early in life. SHRSP and normotensive Wistar-Kyoto (WKY) rats were treated from 6-10 weeks of age with vehicle, hydralazine/hydrochlorothiazide (used as a control for lowered blood pressure) or the angiotensin converting enzyme inhibitor ramipril (3 mg/kg/day). Systolic blood pressures were measured weekly in rats from 6 to 17 weeks of age. In SHRSP (at 17 weeks of age), ramipril-treatment but not hydralazine/hydrochlorothiazide attenuated the long term expression of elevated systolic blood pressure in adult SHRSP while blood pressures of all adult WKY rats were unaffected by any treatment. At 17 weeks, rats were killed and arteries removed for in vitro measurement of isometric contractile activity. Only the incidence of spontaneous oscillations (carotid artery) was affected by ramipril treatment; ramipril did not change the frequency of BayK 8644-induced oscillations in the artery or the frequency of CPA-induced oscillations in aorta from either SHRSP or WKY. These data indicate that while spontaneous oscillations in the carotid artery may be dependent on an angiotensin II-sensitive mechanism during development, agonist-induced oscillations (CPA and BayK 8644) appear not to be angiotensin II-dependent. Thus, not all of the contractile oscillations which appear in vascular smooth muscle from SHRSP are angiotensin II-dependent, suggesting that some of these vascular abnormalities may develop at a time separate from that in which increased blood pressure is firmly established and may not be associated with the for maintenance of elevated blood pressure.

Actions of Norepinephrine and Angiotensin II on Aortic Rings of Adult and Aged Normotensive and Hypertensive Rats

Development of desensitization to physiologic concentration of norepinephrine (NE 10(-9) M) and its effects on angiotensin II (AII) tachyphylaxis in the aortic rings of adult and aged normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) were investigated. Aortic rings of all animals developed desensitization to 10(-9) M NE and tachyphylaxis to AII (10(-9)-10(-6) M). In normotensive WKY, the response to NE and AII was not affected by age, whereas in SHR desensitization to NE and tachyphylaxis to AII were significantly more marked in aged animals. The desensitization to 10(-9) M NE appeared to be homologous because this physiologic concentration of NA significantly attenuated tachyphylaxis to AII (for concentrations of 10(-8)-10(-6) M) in the aortic rings of both adult and aged WKY. Tachyphylaxis to AII was not affected by 10(-9) M NE in either adult or aged SHR, however. These data support our previous finding of similar attenuation of AII tachyphylaxis by NE in aortic rings of normotensive Sprague-Dawley rats, and indicate that absence of such attenuation in SHR; the latter finding could reflect a probable lesion in the interactive pathways of these two vasoconstrictors in hypertensive rats.

CEREBROVASCULAR PROTECTION BY SEQUENTIAL BILATERAL CAROTID ARTERY LIGATION IN AGED SPONTANEOUSLY HYPERTENSIVE RATS

1. Sequential bilateral carotid artery ligation (BCL) separated by a 1 week interval was performed on 5 month spontaneously hypertensive rats (SHR) (i.e. SHRSR-B1/Izm) and the developmental course of hypertension and cerebrovascular lesions in advanced age were analysed as compared with those in age-matched sham-operated controls. 2. Behavioural activity and behavioural reaction to light were also investigated in the above-mentioned SHR, young and adult stroke-prone SHR (i.e. SHRSP-A3/Izm), SHR (i.e. B1/Izm) and Wistar-Kyoto rats (i.e. WKY/Izm). 3. All of the control SHR developed severe hypertension resulting in cerebral stroke with focal oedema due to cerebral haemorrhage and infarction as a result of arterionecrosis 18 months after birth. 4. SHR usually die within a few days of BCL. In the present study, however, they successfully survived without cerebrovascular damage for a long time, although they developed a similar severe hypertension in a significantly shorter period of time (P < 0.05) and showed behavioural abnormalities that were probably due to severe cerebral ischaemia. 5. These experimental results suggest an ischaemic tolerance phenomenon in a hypertensive model that was exposed to mild ischaemic stress by unilateral carotid artery ligation (UCL) before the subsequent induction of severe ischaemia by BCL. The results also suggest that an aggravation of hypertensive cerebrovascular changes due to long-lasting ischaemia after BCL was prevented through a possible cumulative effect of ischaemic stress.