Muscarinic cholinoceptors and choline acetyltransferase activity in the hypothalamus of spontaneously hypertensive rats

To study the role of central cholinergic mechanisms in hypertension, we have determined muscarinic receptors using [³H](-)quinuclidinyl benzilate (QNB) and choline acetyltransferase (ChAT) activity in the brain regions of spontaneously hypertensive rats (SHR), stroke-prone SHR (SHRSP) and renal hypertensive rats. The number of muscarinic receptors was significantly (33–38%) elevated in the hypothalamus of SHR and SHRSP at the ages of 16 and 24 weeks compared to that of Wistar-Kyoto rats (WKY). An increased density of muscarinic receptors was consistently observed in the prehypertensive (5 weeks) and developmental (10 weeks) stages of hypertension. In contrast, in the hypothalamus of rats with renal hypertension there was no muscarinic receptor alteration. The receptor alteration in the SHRSP hypothalamus was not abolished by a chronic hypotensive treatment which prevented the development of hypertension, suggesting that an enhancement of the muscarinic receptors in spontaneous hypertension does not occur secondarily to the elevation of blood pressure. The hypothalamus of SHR and SHRSP at the ages of 5 and 24 weeks showed significantly less activity of ChAT. These data demonstrate that there is a specific increase in muscarinic receptors and a decrease in cholinergic activity in the hypothalamus of SHR and SHRSP. Thus, the present study suggests an important role for hypothalamic cholinergic receptors in the pathogenesis of spontaneous hypertension.     

Effects of gamma-2-melanocyte-stimulating hormone on protein kinase C activity and expression in spontaneous hypertensive rats (SHR).

Administration of gamma-2-melanocyte stimulating hormone (gamma-2-MSH) to rats increases blood pressure, heart rate and natriuresis by acting through the nervous system and this response is more pronounced in spontaneous hypertensive rat (SHR). The molecular mechanisms underlying these effects are unknown, however, protein kinase C (PKC) activity is higher in SHR tissues and melanocortins are known to activate the phosphoinositide (PI) signaling pathway. In this study, we tested the hypothesis that gamma-2-MSH potentiation of PKC activation is increased in nerve terminals from SHR brain. Synaptosomes were isolated from SHR and age-matched control Wistar Kyoto (WKY) rats and incubated with gamma-2-MSH. Total particulate-fraction associated PKC activity was determined and the expression of individual isozymes analyzed by immunoblotting. Treatment with gamma-2-MSH resulted in an increase in particulate-associated PKC activity in hindbrain synaptosomes that was more prominent in SHR. The levels of membrane-associated PKC-alpha and beta-isozymes were considerably less than for PKC-gamma in these tissues as determined by immunoblotting. The novel PKC isozymes delta and epsilon were detected in total synaptosomes but not in membrane fractions. These data suggest that PKC-gamma is the major presynaptic PKC isozyme and that PKC may be an important mediator for gamma-2-MSH in neural tissues.     

Alterations of NO synthase isoforms in brain and kidney of rats with genetic and salt hypertension

Both brain and peripheral nitric oxide (NO) play a role in the control of blood pressure and circulatory homeostasis. Central NO production seems to counteract angiotensin II-induced enhancement of sympathetic tone. The aim of our study was to evaluate NO synthase (NOS) activity and protein expression of its three isoforms--neuronal (nNOS), endothelial NOS (eNOS) and inducible (iNOS)--in two brain regions involved in blood pressure control (diencephalon and brainstem) as well as in the kidney of young adult rats with either genetic (12-week-old SHR) or salt-induced hypertension (8-week-old Dahl rats). We have demonstrated reduced nNOS and iNOS expression in brainstem of both hypertensive models. In SHR this abnormality was accompanied by attenuated NOS activity and was corrected by chronic captopril treatment which prevented the development of genetic hypertension. In salt hypertensive Dahl rats nNOS and iNOS expression was also decreased in the diencephalon where neural structures important for salt hypertension development are located. As far as peripheral NOS activity and expression is concerned, renal eNOS expression was considerably reduced in both genetic and salt-induced hypertension. In conclusions, we disclosed similar changes of NO system in the brainstem (but not in the diencephalon) of rats with genetic and salt-induced hypertension. Decreased nNOS expression was associated with increased blood pressure due to enhanced sympathetic tone.     

Role of intramitochondrial nitric oxide in rat heart and kidney during hypertension

Nitric oxide (NO) is an important reactive molecule in many organisms. A mitochondrial nitric oxide synthase has been described; however, the role of NO in this organelle is not yet fully clear. We tested the effect of intramitochondrial NO on various functions from spontaneously hypertensive rats (SHR) and their normotensive genetic control, Wistar-Kyoto (WKY) rats. While the stimulation of intramitochondrial NOS increased calcium- and phosphate-induced permeability transition pore opening, its inhibition partially prevented it, without affecting membrane potential. Matrix free calcium and the pH decreased with NOS inhibition. Basal [NO] was lower in SHR than in WKY. Our data suggest that intramitochondrial NO plays an important role in mitochondrial regulation during hypertension.     

Antioxidant activity of propionyl-L-carnitine in liver and heart of spontaneously hypertensive rats

Oxidative stress plays an important role in arterial hypertension and propionyl-L-carnitine (PLC) has been found to protect cells from toxic reactive oxygen species. In this work, we have evaluated the antioxidant capacity of chronic PLC treatment in spontaneously hypertensive rats (SHR) by measuring the activity of antioxidant enzymes and the lipid peroxidation in liver and cardiac tissues. The activity of glutathione peroxidase was decreased in liver and cardiac tissues of SHR when compared with their normotensive controls, Wistar- Kyoto (WKY) rats, this alteration being prevented by PLC treatment. Glutathione reductase activity was increased in hypertensive rats and no effect was observed after the treatment. No significant changes in superoxide dismutase activity were observed among all experimental groups. Liver of hypertensive rats showed higher catalase activity than that of normotensive rats, and PLC enhanced this activity in both rat strains. Thiobarbituric acid reactive substances, determined as a measure of lipid peroxidation, were increased in SHR compared with WKY rats, and PLC treatment decreased these values not only in hypertensive rats but also in normotensive ones. The content of carnitine in serum, liver and heart was higher in PLC-treated rats, but PLC did not prevent the hypertension development in young SHR. In addition, triglyceride levels, which were lower in SHR than WKY rats, were reduced by chronic PLC treatment in both rat strains. These results demonstrate: i) the hypotriglyceridemic effect of PLC and ii) the antioxidant capacity of PLC in SHR and its beneficial use protecting tissues from hypertension-accompanying oxidative damage.     

Elastase-like enzyme in the aorta of spontaneously hypertensive rats

In an attempt to obtain information regarding vascular elastase in arterial hypertension, we examined biochemical changes in elastase-like enzyme activity, and the intravascular localization of elastase by immunohistochemical techniques in the aorta of spontaneously hypertensive rats (SHR). In the biochemical study, aortic elastase-like enzyme activity was significantly higher in SHR than in controls. Using an antibody against rat pancreatic elastase raised in the rabbit, it was demonstrated immunohistochemically that the enzyme was localized in the endothelial cells and subendothelial spaces in the aorta of control animals. In SHR, elastase was also demonstrated in medial smooth muscle cells and particularly in the modified smooth muscle cells in areas of intimal thickening. Some vacuoles in the smooth muscle cells also showed positive enzyme staining. Elastase seems to play an important role in the development of hypertensive vascular changes.     

Relative contribution of Rho kinase and protein kinase C to myogenic tone in rat cerebral arteries in hypertension

Arterial smooth muscle constriction in response to pressure, i.e., myogenic tone, may involve calcium-dependent and calcium-sensitization mechanisms. Calcium sensitization in vascular smooth muscle is regulated by kinases such as PKC and Rho kinase, and activity of these kinases is known to be altered in cardiovascular disorders. In the present study, we evaluated the relative contribution of PKC and Rho kinase to myogenic tone in cerebral arteries in hypertension. Myogenic tone and arterial wall calcium in Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) were measured simultaneously, and the effect of PKC and Rho kinase inhibitors on myogenic tone was evaluated. SHR arteries showed significantly greater myogenic tone than WKY arteries. Pressure/wall tension-arterial wall calcium curves showed a hyperbolic relation in WKY rats, but the curves for SHR arteries were parabolic. Myogenic tone was decreased by the Rho kinase inhibitors Y-27632 and HA-1077, with a significantly greater effect in SHR than in WKY arteries. Reduction in myogenic tone produced by the PKC inhibitor bisindolylmaleimide I in WKY and SHR arteries was significantly less than that produced by Rho kinase inhibition. The pressure-dependent increase in myogenic tone was significantly decreased by Y-27632, and the decrease was markedly greater than that produced by bisindolylmaleimide I in SHR arteries. In WKY arteries, the pressure-dependent increase in myogenic tone was decreased to a similar extent by Y-27632 and bisindolylmaleimide I. These results suggest greater myogenic tone with increased calcium sensitization in SHR arteries, largely because of Rho kinase activation, with a minor contribution of PKC activation.     

ARHGAP21 associates with FAK and PKCzeta and is redistributed after cardiac pressure overload

ARHGAP21 is highly expressed in the heart, which demonstrates activity over Cdc42 and interacts with proteins of the cytoskeleton and adherent junctions. The main cause of cardiac hypertrophy is mechanical stimulus; therefore we analyzed ARHGAP21 expression after acute mechanical stress in the myocardium and its association with FAK and PKCζ. We demonstrated that ARHGAP21 is relocated to Z-lines and costameres after pressure overload, and interacts with PKCζ and FAK in control rats (sham), rats submitted to aortic clamping and spontaneously hypertensive rats (SHR). Co-transfection using ARHGAP21 and PKCζ constructions demonstrated that ARHGAP21 associates with PKCζ-GST and endogenous FAK. Pulldown assay showed that ARHGAP21 binds to the C-terminal region of FAK. Moreover, ARHGAP21 binds to PKCζ phosphorylated on Thr410 in sham and SHR. However, ARHGAP21 only binds to FAK phosphorylated on Tyr925 of SHR. Additionally, PKCζ is phosphorylated by mechanical stimuli. These results suggest that ARHGAP21 may act as a signaling or scaffold protein of FAK and PKCζ signaling pathways, developing an important function during cardiac stress.     

Gender-related distinctions in protein kinase C activity in rat vascular smooth muscle

Gender differences in vascular reactivity have been suggested; however, the cellular mechanisms involved are unclear. We tested the hypothesis that the gender differences in vascular reactivity reflect gender-related, possibly estrogen-mediated, distinctions in the expression and activity of specific protein kinase C (PKC) isoforms in vascular smooth muscle. Aortic strips were isolated from intact and gonadectomized male and female Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). Isometric contraction was measured in endothelium-denuded aortic strips. PKC activity was measured in the cytosolic and particulate fractions, and the amount of PKC was measured using Western blots and isoform-specific anti-PKC antibodies. In intact male WKY rats, phenylephrine (Phe, 10(-5) M) and phorbol 12,13-dibutyrate (PDBu, 10(-6) M) stimulated contraction to 0.37 +/- 0.02 and 0.42 +/- 0.02 g/mg tissue wt, respectively. The basal particulate/cytosolic PKC activity ratio was 0.86 +/- 0.06, and Western blots revealed alpha-, delta-, and zeta-PKC isoforms. Phe and PDBu increased PKC activity and caused significant translocation of alpha- and delta-PKC from the cytosolic to particulate fraction. In intact female WKY rats, basal PKC activity, the amount of alpha-, delta-, and zeta-PKC, the Phe- and PDBu-induced contraction, and PKC activity and translocation of alpha- and delta-PKC were significantly reduced compared with intact male WKY rats. The basal PKC activity, the amount of alpha-, delta-, and zeta-PKC, the Phe and PDBu contraction, and PKC activity and alpha- and delta-PKC translocation were greater in SHR than WKY rats. The reduction in Phe and PDBu contraction and PKC activity in intact females compared with intact males was greater in SHR ( approximately 30%) than WKY rats ( approximately 20%). Phe and PDBu contraction and PKC activity were not significantly different between castrated males and intact males but were greater in ovariectomized (OVX) females than intact females. Treatment of OVX females or castrated males with 17 beta-estradiol, but not 17 alpha-estradiol, subcutaneous implants caused significant reduction in Phe and PDBu contraction and PKC activity that was greater in SHR than WKY rats. Phe and PDBu contraction and PKC activity in OVX females or castrated males treated with 17 beta-estradiol plus the estrogen receptor antagonist ICI-182,780 were not significantly different from untreated OVX females or castrated males. Thus a gender-related reduction in vascular smooth muscle contraction in female WKY rats with intact gonads compared with males is associated with reduction in the expression and activity of vascular alpha-, delta-, and zeta-PKC. The gender differences in vascular smooth muscle contraction and PKC activity are augmented in the SHR and are possibly mediated by estrogen.     

In situ analysis of microvascular pericytes in hypertensive rat brains

We used immunofluorescence microscopy and isoactin-specific antibodies to characterize the pattern and prevalence of pericytes within the brain microcirculation. Blood pressures of normotensive, Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats were measured prior to sacrifice and pressure-perfusion fixation. WKY and SHR brains were subdivided into ten major regions prior to ultracryomicrotomy. Sections 0.3-0.5 micron wide were treated with 10-40 micrograms/ml affinity-purified antibodies to the muscle and non-muscle actin isoforms. These localization studies show that there are four times the number of pericyte-rich capillaries in the SHR motor cortex compared to WKY counterparts (59.9 vs. 15.3%). In contrast, the sensory cortex of both rat strains is deficient in muscle actin staining surrounding the capillaries. The most striking difference in pericyte presence and muscle actin antibody staining between the SHR and WKY was observed in the tegmentum of the brainstem. There is nearly a one-to-one coincidence observed in pericyte and capillary profiles present within thin, frozen sections of the SHR midbrain. SHR pons capillaries were also pericyte-enriched. WKY analyses of plastic embedded thin sections confirmed the presence of pericytes and their filament-enriched processes encircling the capillaries of the hypertensive brains. These results suggest that pericytes may play important roles in hypertension and cerebrovascular disease processes.     

Glutathione system in young spontaneously hypertensive rats

Glutathione (GSH) forms a part of the antioxidant system that plays a vital role in preventing oxidative stress, and an imbalance in the oxidant/antioxidant system has been linked to the pathogenesis of hypertension. The aim of this study was to investigate the status of the GSH system in the kidney of spontaneously hypertensive rats (SHR). Components of the GSH system, including glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-S-transferase (GST), and total GSH content, were measured in the kidneys of 4, 6, 8, 12, and 16 weeks old SHR and Wistar–Kyoto (WKY) rats. Systolic blood pressure of SHR was significantly higher from the age of 6 weeks onwards compared with age-matched WKY rats. GPx activity in the SHR was significantly lower from the age of 8 weeks onwards when compared to that in age-matched WKY rats. No significant differences were evident in the GPx-1 protein abundance, and its relative mRNA levels, GR, GST activity, and total GSH content between SHR and age-matched WKY rats. The lower GPx activity suggests of an impairment of the GSH system in the SHR, which might be due to an abnormality in its protein rather than non-availability of a cofactor. Its role in the development of hypertension in SHR however remains unclear.     

Sleep-related sympathovagal imbalance in SHR

The role of the autonomic nervous system in spontaneous hypertension during each stage of the sleep-wake cycle remains unclear. The present study attempted to evaluate the differences in cardiac autonomic modulations among spontaneously hypertensive rats (SHR), normotensive Wistar-Kyoto rats (WKY), and Sprague-Dawley rats (SD) across sleep-wake cycles. Continuous power spectral analysis of electroencephalogram, electromyogram, and heart rate variability was performed in unanesthetized free moving rats during daytime sleep. Frequency-domain analysis of the stationary R-R intervals (RR) was performed to quantify the high-frequency power (HF), low-frequency power (LF)-to-HF ratio (LF/HF), and normalized LF (LF%) of heart rate variability. WKY and SD had similar mean arterial pressure, which is significantly lower than that of SHR during active waking, quiet sleep, and paradoxical sleep. Compared with WKY and SD, SHR had lower HF but similar RR, LF/HF, and LF% during active waking. During quiet sleep, SHR developed higher LF/HF and LF% in addition to lower HF. SHR ultimately exhibited significantly lower RR accompanied with higher LF/HF and LF% and lower HF during paradoxical sleep compared with WKY. We concluded that significant cardiac sympathovagal imbalance with an increased sympathetic modulation occurred in SHR during sleep, although it was less evident during waking.     

Functional and structural pattern of arterial responses in hereditary hypertriglyceridemic and spontaneously hypertensive rats in early stage of experimental hypertension

It has been shown that endothelium-derived nitric oxide (NO) plays an important role in regulation of vascular tone in the prenatal and early postnatal period. The aim of this paper was to determine the reactivity and accompanying structural changes in thoracic aorta from 4-week-old spontaneously hypertensive rats (SHR) and rats with hereditary hypertriglyceridemia (hHTG) in comparison with age-matched normotensive controls. For functional studies thoracic aorta was excised, cut into rings and mounted in organ baths for measurement of isometric contractile force. For morphological studies cardiovascular system of rats was perfused with glutaraldehyde fixative (at 100 mm Hg) via cannula placed in the left ventricle. Morphological changes of thoracic aorta were measured using light microscopy. Systolic blood pressure (SBP) in SHR (98+/-1 mm Hg) did not significantly differ from that of age-matched control rats (95+/-4 mm Hg), but was slightly increased in hHTG rats (110+/-2 mm Hg, P<0.05). Heart weight/body weight ratio was higher in SHR and hHTG rats than in control group indicating the hypertrophy of the heart in both models of hypertension. Endothelium-dependent relaxation of aorta induced by acetylcholine was preserved in all groups and did not differ from that in control normotensive rats. The maximal isometric contraction of thoracic aorta to noradrenaline (NA) was reduced in hypertensive groups and the concentration-response curves to NA were shifted to the right indicating increased sensitivity of smooth muscle to NA. The values of wall thickness and cross sectional area as well as inner diameter of thoracic aorta in SHR and hHTG rats were significantly decreased in comparison to control groups. Endothelial dysfunction seems to be absent in all young rats before development of hypertension. In conclusion, our observations indicate that in early stage of experimental hypertension NO-dependent relaxation is preserved so that putative impairment of this function provides no significant pathogenic contribution to the onset of hypertension in these two experimental models.     

Somatosympathetic reflex in rats with various models of arterial hypertension

Spontaneous and reflex activities of sympathetic nerve were compared in animals with arterial hypertension of different aetiology. Reflex discharges elicited by single-shock stimulation of afferent fibres were recorded. In acute experiences on anaesthetized rats with renovascular and spontaneous (SHR) model of arterial hypertension, electric basal and evoked activity (somatosympathetic reflex) in cervical sympathetic trunk were recorded. It is shown, that the spontaneous electric activity in sympathetic nerve of hypertensive rats is larger than spontaneous activity of normotensive control animals. The somatosympathetic reflex in hypertensive rats differs from that of control animals. In rats with renovascular model of hypertension, the reflex magnitude is reduced, and in the SHR the reflex is increased. Time characteristics of the reflex in hypertensive rats differed among them. It is suggested that functional activities of the brain stem in rats with different arterial hypertension model are unequal.     

Inhibition of neurons in commissural nucleus of solitary tract reduces sympathetic nerve activity in SHR

Neurons in the commissural nucleus of the solitary tract (commNTS) play an important role in certain cardiovascular responses dependent on sympathetic vasoconstrictor activation, including the arterial chemoreceptor reflex. Electrolytic lesions of the commNTS elicit a fall in arterial pressure (AP) in spontaneously hypertensive rats (SHR). To determine whether the latter result 1) arose from elimination of commNTS neuronal activity rather than en passant axons and 2) was accompanied by a reduction in sympathetic nerve activity, we evaluated the effect of inhibition of neurons in the commNTS on basal splanchnic sympathetic nerve activity (SNA), AP, and heart rate (HR) in SHR, Wistar-Kyoto (WKY), and Sprague-Dawley (SD) rats. In chloralose-anesthetized, paralyzed, and artificially ventilated SHR, microinjection of GABA into the commNTS markedly decreased splanchnic SNA, AP, and HR. The reductions in SNA and AP following similar microinjections in WKY and SD rats were significantly less than those in SHR. Our findings suggest that tonically active neurons in the commNTS contribute to the maintenance of SNA and the hypertension in SHR. The level of tonic discharge of these commNTS neurons in normotensive WKY and SD rats may be lower than in SHR.     

The Orexin System and Hypertension

In this review, we focus on the role of orexin signaling in blood pressure control and its potential link to hypertension by summarizing evidence from several experimental animal models of hypertension. Studies using the spontaneously hypertensive rat (SHR) animal model of human essential hypertension show that pharmacological blockade of orexin receptors reduces blood pressure in SHRs but not in Wistar–Kyoto rats. In addition, increased activity of the orexin system contributes to elevated blood pressure and sympathetic nerve activity (SNA) in dark-active period Schlager hypertensive (BPH/2J) mice, another genetic model of neurogenic hypertension. Similar to these two models, Sprague-Dawley rats with stress-induced hypertension display an overactive central orexin system. Furthermore, upregulation of the orexin receptor 1 increases firing of hypothalamic paraventricular nucleus neurons, augments SNA, and contributes to hypertension in the obese Zucker rat, an animal model of obesity-related hypertension. Finally, we propose a hypothesis for the implication of the orexin system in salt-sensitive hypertension. All of this evidence, coupled with the important role of elevated SNA in increasing blood pressure, strongly suggests that hyperactivity of the orexin system contributes to hypertension.     

Proliferation of thyrotropin releasing hormone receptors in specific brain regions during the development of hypertension in spontaneously hypertensive rats

The binding of [3H] [3-MeHis2] thyrotropin releasing hormone [( 3H]MeTRH) to brain membranes prepared from 8 week old spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats was determined. [3H]MeTRH bound specifically to rat brain membranes at a single high affinity site. The density (Bmax value) of [3H]MeTRH binding sites was significantly greater (28%) in SHR rats compared to WKY rats. The apparent dissociation constants (Kd values) for the binding of [3H]MeTRH in SHR and WKY rats did not differ. Binding in the various brain regions revealed that the density of [3H]MeTRH was highest in the hypothalamus followed in decreasing order by pons + medulla, midbrain, cortex and striatum. The binding of [3H]MeTRH was approximately 25% greater in cortex, hypothalamus and striatum of SHR rats in comparison to WKY rats. The binding in pons + medulla, midbrain and pituitary of SHR and WKY rats did not differ. To assess the significance of increased binding sites for [3H]MeTRH in some brain regions of SHR rats, the binding studies were carried out during normotensive and hypertensive stages of postnatal age in the two strains. In 3 and 4 week old SHR rats there was neither an increase in blood pressure nor any increase in [3H]MeTRH binding in the hypothalamus and striatum as compared to age matched WKY rats. With the development of elevated blood pressure at 6 weeks, an increase in [3H]MeTRH binding in the hypothalamus and striatum of SHR rats in comparison to the tissues from WKY rats was observed. The results provide, for the first time, evidence for a parallel increase in the density of brain TRH receptors with elevation of blood pressure, and suggest that brain TRH receptors may play an important role in the pathophysiology of hypertension.     

Increased plasma level of asymmetric dimethylarginine in hypertensive rats facilitates platelet aggregation: role of plasma tissue factor

This study was designed to explore the role of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthesis, in platelet aggregation in hypertension and its possible mechanisms. Spontaneously hypertensive rats (SHR) and L-NAME-induced hypertensive rats were orally administered with L-arginine (1 g/(kg·day) for 14 days. Systolic blood pressure, platelet aggregation, and plasma tissue factor (TF) level and activity were measured. The plasma concentration of ADMA in SHR was determined. In vitro, platelet-rich plasma isolated from Wistar rats was prepared in order to observe the effect of exogenous ADMA on platelet aggregation and TF level and (or) activity in platelet-rich plasma. In both types of hypertensive rats, systolic blood pressure, platelet aggregation, and the level and activity of plasma TF were elevated compared with corresponding control animals. Plasma ADMA level was also increased in SHR. Treatment with L-arginine, a competitor of ADMA, lowered blood pressure and inhibited platelet aggregation concomitantly with a decrease in plasma TF level and activity in both types of hypertensive rats. We also found that exogenous ADMA promoted platelet aggregation and increased TF level and (or) activity in platelet-rich plasma, an effect that was inhibited by pretreatment with L-arginine. Importantly, the enhanced platelet aggregation induced by exogenous ADMA was reduced by pretreatment with anti-TF antibody. The results suggest that endogenous ADMA may be involved in platelet hyperaggregation status in hypertension, and the facilitation of platelet aggregation by ADMA is related to upregulation of the level and activity of plasma TF.     

The participation of brain NO synthase in blood pressure control of adult spontaneously hypertensive rats

Increased blood pressure (BP) in genetic hypertension is usually caused by high activity of sympathetic nervous system (SNS) which is enhanced by central angiotensin II but lowered by central nitric oxide (NO). We have therefore evaluated NO synthase (NOS) activity as well as neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS) protein expression in brainstem and midbrain of adult spontaneously hypertensive rats (SHR) characterized by enhanced sympathetic vasoconstriction. We also studied possible participation of brain NO in antihypertensive effects of chronic captopril treatment of adult SHR. NOS activity was increased in midbrain of SHR compared to Wistar-Kyoto (WKY) rats. This could be ascribed to enhanced iNOS expression, whereas nNOS expression was unchanged and eNOS expression was reduced in this brain region. In contrast, no significant changes of NOS activity were found in brainstem of SHR in which nNOS and iNOS expression was unchanged, but eNOS expression was increased. Chronic captopril administration lowered BP of adult SHR mainly by attenuation of sympathetic tone, whereas the reduction of angiotensin II-dependent vasoconstriction and the decrease of residual BP (amelioration of structural remodeling of resistance vessels) were less important. This treatment did not affect significantly either NOS activity or expression of any NOS isoform in the two brain regions. Our data do not support the hypothesis that altered brain NO formation contributes to sympathetic hyperactivity and high BP of adult SHR with established hypertension.     

B-50/GAP-43 Phosphorylation and PKC Activity are Increased in Rat Hippocampal Synaptosomal Membranes After an Inhibitory Avoidance Training

Several lines of evidence indicate that protein kinase C (PKC) is involved in long-term potentiation (LTP) and in certain forms of learning. Recently, we found a learning-specific, time-dependent increase in [³H]phorbol dibutyrate binding to membrane-associated PKC in the hippocampus of rats subjected to an inhibitory avoidance task. Here we confirm and extend this observation, describing that a one trial inhibitory avoidance learning was associated with rapid and specific increases in B-50/GAP-43 phosphorylation in vitro and in PKC activity in hippocampal synaptosomal membranes. The increased phosphorylation of B-50/GAP-43 was seen at 30 min (+35% relative to naive or shocked control groups), but not at 10 or 60 min after training. This learning-associated increase in the phosphorylation of B-50/GAP-43 is mainly due to an increase in the activity of PKC. This is based on three different sets of data: 1) PKC activity increased by 24% in hippocampal synaptosomal membranes of rats sacrificed 30 min after training; 2) B-50/GAP-43 immunoblots revealed no changes in the amount of this protein among the different experimental groups; 3) phosphorylation assays, performed in the presence of bovine purified PKC or in the presence of the selective PKC inhibitor CGP 41231, exhibited no differences in B-50/GAP-43 phosphorylation between naive and trained animals. In conclusion, these results support the contention that hippocampal PKC participates in the early neural events of memory formation of an aversively-motivated learning task.