Presence of angiotensin II in neurons cultured from fetal rat brain

By means of a specific antiserum to angiotensin II (ANGII), it has been possible to demonstrate the existence of ANGII immunoreactivity in cells cultured from fetal rat brain. Dissociated cells cultured from the whole brains of 20-day-old fetuses were stained according to the peroxidase antiperoxidase method. Immunoreactive ANGII was observed in less than 1% of the total cell population. These cells have been identified as neurons on the basis of morphological criteria. Positively stained neuronal processes with localized concentrations of ANGII immunoreactivity were visualized in association with ANGII-containing soma. The presence of ANGII in cultured brain cells provides an experimental model for investigating the role of angiotensin in the CNS.     

PI3-kinase inhibitors abolish the enhanced chronotropic effects of angiotensin II in spontaneously hypertensive rat brain neurons

Angiotensin II (Ang II), acting at Ang II type 1 receptors (AT1Rs), increases the firing rate of neurons from Wistar-Kyoto (WKY) rat brain via protein kinase C (PKC)- and calcium-calmodulin kinase II (CaMKII)-dependent mechanisms. The objectives of this study were twofold; first, to compare the Ang-II-stimulated increase in firing of neurons from WKY and spontaneous hypertensive rats (SHR) and second, to elucidate the signaling mechanisms involved. Action potentials were measured in neurons cultured from SHR and WKY rat brains using the whole cell configuration of the patch-clamp technique in the current-clamp mode. Ang II (100 nM) caused three- and sixfold increases in neuronal firing rate in WKY rat and SHR neurons, respectively; effects that were abolished by the AT1R antagonist Losartan (1 microM). Co-administration of calphostin C (10 microM, a PKC inhibitor) and KN-93 (10 microM, a CaMKII inhibitor) completely blocked this Ang II action in WKY rat neurons, while they caused only a approximately 50% attenuation in SHR neurons. The residual increase in firing rate produced by Ang II in SHR neurons was blocked by inhibitors of phosphatidylinositol 3 kinase (PI3-kinase), either LY 294002 (10 microM) or wortmannin (100 nM). These observations suggest that a PI3-kinase signaling pathway may be responsible for the enhanced chronotropic effect produced by Ang II in SHR neurons.     

Chronotropic effect of angiotensin II via type 2 receptors in rat brain neurons

Previously, we determined that angiotensin II (Ang II) elicits an Ang II type 2 (AT(2)) receptor-mediated increase of neuronal delayed rectifier K(+) (I(KV)) current in neuronal cultures from newborn rat hypothalamus and brain stem. This requires generation of lipoxygenase (LO) metabolites of arachidonic acid (AA) and activation of serine/threonine phosphatase type 2A (PP-2A). Enhancement of I(KV) results in a decrease in net inward current during the action potential (AP) upstroke as well as shortening of the refractory period, which may lead to alterations in neuronal firing rate. Thus, in the present study, we used whole-cell current clamp recording methods to investigate the AT(2) receptor-mediated effects of Ang II on the firing rate of cultured neurons from the hypothalamus and brain stem. At room temperature, these neurons exhibited spontaneous APs with an amplitude of 77.72 +/- 2.7 mV (n = 20) and they fired at a frequency of 0.8 +/- 0.1 Hz (n = 11). Most cells had a prolonged early after-depolarization that followed an initial fully developed AP. Superfusion of Ang II (100 nM) plus losartan (LOS, 1 microM) to block Ang II type 1 receptors elicited a significant chronotropic effect that was reversed by the AT(2) receptor inhibitor PD 123,319 (1 microM). LOS alone had no effect on any of the parameters measured. The chronotropic effect of Ang II was reversed by the general LO inhibitor 5,8,11,14-eicosatetraynoic acid (10 microM) or by the selective PP-2A inhibitor okadaic acid (1 nM) and was mimicked by the 12-LO metabolite of AA 12-(S)-hydroxy-(5Z, 8Z, 10E, 14Z)-eicosatetraynoic acid. These data indicate that Ang II elicits an AT(2) receptor-mediated increase in neuronal firing rate, an effect that involves generation of LO metabolites of AA and activation of PP-2A.     

Stress and brain angiotensin II receptors.

Angiotensin II (ANG II) has been recognized recently as one of the stress hormones that participate in various stress-induced responses, including sympathetic (pressor, tachycardiac, and hyperthermic) and neuroendocrine responses. Brain ANG II receptors have been identified in many brain regions involved in the stress responses. During stress-exposure, ANG II increases in the plasma and in the brain. Central administration of ANG II induces stress responses, whereas central blockade of ANG II receptors results in the inhibition of the sympathetic and neuroendocrine responses to stress. All these findings indicate that ANG II and its receptors contribute to the development of various sympathetic and neuroendocrine responses during stress exposure. This review focuses on the role of brain ANG II receptors in the stress-induced responses.     

Direct action of angiotensin II on central neurons

Response of nerve cells of the somatosensory and visual cortex and anterior hypothalamus of rabbits and also of the isolated circumesophageal nerve ring ofHelix pomatia to microiontophoretic administration of angiotensin II (A-II) were studied. Responses of the rabbit brain neurons to A-II were characterized by a marked increase in the frequency of spike discharges which depended on the dose of the drug injected. Neurons of the anterior hypothalamus had a lower threshold of response than cortical neurons. Application of A-II to the soma of the recorded snail cells caused a marked but reversible decrease in the membrane potential level. The resistance of the membrane under these circumstances was reduced by two to four times. These results are evidence of the direct action of A-II on central neurons.P. K. Anokhim Research Institute of Normal Physiology, Acadamy of Medical Sciences of the USSR, Moscow. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol1. 82, No. 8, pp. 899–902, August, 1976.     

Modulation by endothelium of the vascular effects of angiotensin II

Modulation by vascular endothelium of the effects of AII was studied in the isolated rabbit aortic and superior mesenteric artery strips. The contractile effect of AII was enhanced in rubbed aortic strips. Similar enhancement was obtained in hydroquinone pretreated unrubbed strips. The relaxing effect of acetylcholine in AII-induced precontracted aortic strips was abolished after rubbing and hydroquinone pretreatment. However, no significant changes were observed in the contractile response to AII on aspirin and nicotine pretreated strips.In the isolated mesenteric artery strips AII produced a biphasic responses. The contractile effect of AII was enhanced in rubbed strips. Similar potentiation was also obtained in hydroquinone, aspirin and nicotine pretreated unrubbed strips. The relaxation phase of AII response was completely abolished in rubbed strips but partially inhibited in hydroquinone, aspirin and nicotine preatreated unrubbed strips.From these results it was concluded that EDRF is the main endothelial humoral factor which modulates the effect of AII in the rabbit aorta while both EDRF and PGI₂ are involved for the modulation of the effects of octapeptide in the mesenteric artery.This work was supported by a grant from Turkish Scientific and Technical Research Council (TAG-578).     

Activation of subthalamic neurons by contralateral subthalamic deep brain stimulation in Parkinson disease

Multiple studies have shown bilateral improvement in motor symptoms in Parkinson disease (PD) following unilateral deep brain stimulation (DBS) of the subthalamic nucleus (STN) and internal segment of the globus pallidus, yet the mechanism(s) underlying this phenomenon are poorly understood. We hypothesized that STN neuronal activity is altered by contralateral STN DBS. This hypothesis was tested intraoperatively in humans with advanced PD using microelectrode recordings of the STN during contralateral STN DBS. We demonstrate alterations in the discharge pattern of STN neurons in response to contralateral STN DBS including short latency, temporally precise, stimulation frequency-independent responses consistent with antidromic activation. Furthermore, the total discharge frequency during contralateral high frequency stimulation (160 Hz) was greater than during low frequency stimulation (30 Hz) and the resting state. These findings demonstrate complex responses to DBS and imply that output activation throughout the basal ganglia-thalamic-cortical network rather than local inhibition is a therapeutic mechanism of DBS.     

Modulation of a delayed-rectifier K+ current by angiotensin II in rat sympathetic neurons

It is well known that angiotensin II (Angio II) mimics most of the muscarinic-mediated excitatory actions of acetylcholine on superior cervical ganglion neurons. For instance, in addition to depolarization and stimulation of norepinephrine release, muscarinic agonists and Angio II modulate the M-type K(+) current and the N-type Ca(2+) current. We recently found that muscarinic receptors modulate the delayed rectifier current I(KV) as well. Therefore a whole cell patch-clamp experiment was carried out in rat cultured sympathetic neurons to assess whether Angio II modulates I(KV). We found that Angio II increased I(KV) by about 30% with a time constant of approximately 30 s. In comparison, inhibition of M-current was faster (tau approximately 8 s) and stronger ( approximately 61%). Modulation of I(KV) was disrupted by the AT(1) receptor-antagonist losartan but not by the AT(2)-antagonist PD123319. I(KV) enhancement was reduced by the G-protein inhibitor GDP-beta-S, whereas current modulation remained unaltered after cell treatment with pertussis toxin. The peptidergic modulation of I(KV) was severely disrupted when internal ATP was replaced by its nonhydrolyzable analogue AMP-PNP. Angio II enhanced I(KV) and further reduced the stimulatory action of a muscarinic agonist on I(KV). Likewise, the muscarinc agonist enhanced I(KV) and occluded the effect of Angio II on I(KV). We have also found that the protein kinase C activator PMA enhanced I(KV), thereby mimicking and further attenuating the action of Angio II on I(KV). These results suggest that AT(1) receptors by coupling to pertussis toxin-insensitive G proteins, stimulate an ATP-dependent and PKC-mediated pathway to modulate I(KV).     

A comparison between the prostaglandin releasing effects of angiotensin II and angiotensin III

Angiotensin II and its natural fragment (des-aspartic acid)¹-angiotensin II (angiotensin III) induced a dose-dependent contraction in the isolated rat stomach fundus strip and rat colon. 1-Acetyl-2-(8-chloro-10,11-dihydrodibenz(b,f)(1,4)oxazepine-10, carbonyl) hydrazine (SC 19220), a widely used competitive blocker of prostaglandins and acetyl salicyclic acid, a well-known inhibitor of prostaglandin biosynthesis, partially abolished the contraction induced by both peptides in the rat stomach fundus but not in the rat colon. The inhibition induced by SC 19220 and acetyl salicyclic acid was found to be higher for angiotensin III than angiotensin II when the dose-response curves and equipotent concentrations of the peptides were compared before and after the drugs.These results were taken as evidence that some component of the contractile effects of angiotensin II and angiotensin III on the isolated rat stomach fundus involves the release of prastaglandins by the peptides and in this respect angiotensin III has higher potency than angiotensin II.This work is supported by a grant from the Turkish Scientific and Technical Research Council (TAG-350).     

Signalling across the blood brain barrier by angiotensin II: novel implications for neurogenic hypertension

Angiotensin II (AngII) is a major culprit in essential hypertension. Based on a genetic rodent model of hypertension, we review here evidence that AngII may signal across the blood brain barrier to affect neuronal circuits within the nucleus tractus solitarii (NTS) of the brainstem, a pivotal region regulating both the baroreceptor reflex and set point control of arterial pressure. We have termed this form of signalling as vascular–neuronal signalling. We describe that the depressant action of AngII in NTS on the baroreceptor reflex is mediated via activation of endothelial nitric oxide synthase (eNOS) releasing NO that promotes release of the inhibitory transmitter—GABA. This could shunt the incoming excitatory baroreceptor afferent traffic impinging on NTS neurones. Chronic studies recording arterial pressure in conscious unrestrained rats using radio-telemetry have revealed that eNOS in NTS plays an endogenous physiological role in the homeostatic regulation of the gain of the cardiac baroreceptor reflex. However, in the spontaneously hypertensive rat, eNOS mRNA was higher (compared to normotensive rats), and its chronic blockade in NTS restored the abnormally depressed cardiac baroreceptor reflex to levels akin to normotensive rats, improved heart rate variability and lowered arterial pressure. Hence, it seems that excessive eNOS activity in NTS of the SHR contributes to the pathological state of this animal model’s cardiovascular autonomic nervous system. We speculate on why eNOS activity may be up regulated in the NTS of the SHR and propose that it is a consequence of high cerebral vascular resistance and inadequate blood perfusion of the brainstem.     

Acute reversal by saralasin of multiple intrarenal effects of angiotensin II.

Glomerular hemodynamics were measured by micropuncture technique in the plasma volume-expanded Munich-Wistar rat in 1) a control group, 2) during a pressor infusion of angiotensin II (AII), and 3) during simultaneous infusions of AII and saralasin, which returned arterial pressure to normal. Respective values obtained in the three groups studied were: nephron filtration rate: 60 +/- 2 vs. 40 +/- 2 vs. 42 +/- 2 nl.min-1.g kidney wt-1; nepphron plasma flow: 263 +/- 13 vs. 106 +/- 5 vs. 165 +/- 13 nl. min-1.g kidney wt-1; LpA, the glomerular permeability coefficient: 0.090 +/- 0.009 vs. 0.033 +/- 0.005 vs. 0.103 +/- 0.020 nl.s-1.g kidney wt-1. mmHg-1; afferent arteriolar resistance: 10.2 +/- 0.7 vs. 25.1 +/- 1.3 vs. 19.7 +/- 3.3 10(9) dyn.s.cm-5; efferent arteriolar resistance: 7.8 +/- 0.5 vs. 22.0 +/- 0.9 vs. 10.8 +/- 1.7 10(9) dyn.s.cm-5. Saralasin acutely reversed the effect of AII on both efferent resistance and LpA, suggesting that AII does not decrease LpA by inducing a fixed anatomic change. For unclear reasons, saralasin did not reverse the increase in afferent resistance associated with infusion of AII. Saralasin infusion in high AII states may acutely affect glomerular hemodynamics by decreasing efferent resistance and increasing the glomerular permeability coefficient.     

Pressure-independent cardiac effects of angiotensin II in pigs

BACKGROUND: Angiotensin II (Ang II) is a potent vasoconstrictor with an important role in the development of cardiovascular disease. Earlier results have shown a positive acute inotropic effect of Ang II in anaesthetized pigs together with significant vasoconstriction. This investigation was designed to study cardiac effects of Ang II, when blood pressure was maintained constant by experimental means. METHODS: Ang II (200 microg h(-1)) was infused in anaesthetized pigs (n = 10) at two different arterial blood pressures, the first determined by the effects of Ang II alone, and the second maintained at baseline blood pressure with nitroprusside. Cardiac systolic and diastolic function was evaluated by analysis of left ventricular pressure-volume relationships. RESULTS: Heart rate, end-systolic elastance (Ees) and pre-load adjusted maximal power (PWRmax EDV(-2)) increased at both blood pressure levels, although less when blood pressure was kept constant with nitroprusside. The time constant for isovolumetric relaxation (tau(1/2)) was prolonged with Ang II alone and shortened with Ang II infused together with nitroprusside. CONCLUSION: Ang II infusion in the pig has inotropic and chronotropic properties independent of arterial blood pressure levels, although the effects seem to be blunted by pharmacological actions of the nitric oxide donor nitroprusside.     

Activation of brain stem neurons by irritant chemical stimulation of the throat assessed by c-fos immunohistochemistry

The method of c-fos immunohistochemistry was used to identify the brain stem distribution of neurons activated following irritant chemical stimulation of the laryngopharyngeal mucosa. In pentobarbital-anesthetized rats, either water (control), nicotine (600 mM, 1 ml) or capsaicin (330 μM, 1 ml) was applied to the pharynx via a cannula placed posterior to the soft palate. Following nicotine and capsaicin, there was a significant increase in fos-like immunoreactivity (FLI) compared with controls in the following areas: nucleus of the solitary tract from the level of the pyramidal decussation caudally to the level of the area postrema rostrally; dorsomedial aspect of trigeminal subnucleus caudalis (Vc); and paratrigeminal islands interspersed in the spinal trigeminal tract. There was significantly more FLI in Vc and paratrigeminal nuclei following capsaicin than following nicotine, while the reverse was true for NTS. In addition, there was a significant increase in FLI in area postrema and the ventrolateral medullary region dorsal to the lateral reticular nucleus following nicotine but not capsaicin. The distributions of FLI in NTS, area postrema, Vc, and paratrigeminal nuclei are consistent with prior anatomical tract-tracing studies and suggest roles for these brain stem regions in mediating sensory and reflex responses to irritant chemical stimulation of the upper respiratory mucosa. Electronic Publication     

Renal function in Lophius americanus: effects of angiotensin II.

Arterial blood pressure, urine flow rate, and plasma and urine electrolytes were measured in the aglomerular goosefish (L. americanus) before, during, and after the intravenous infusion of angiotensin II (from 5 to 280 ng/min.kg body wt). Increases in arterial blood pressure were directly related to the logarithm of the angiotensin infusion rate (r = 0.62, P less than 0.005). Angiotensin also increased urine flow from 0.676 +/- 0.065 to 0.755 +/- 0.068 ml/h.kg body wt (P less than 0.005) and Na excretion from 41.0 +/- 5.5 to 54.4 +/- 7.0 mumol/h.kg body wt (P less than 0.001). In 17 of the 19 fish infused with angiotensin the diuretic and natriuretic effects were directly related to the logarithm of the infusion rate (r = 0.44, P less than 0.04 and r = 0.51, P less than 0.02, respectively). There was no relationship between the pressor and the diuretic or natriuretic effects of angiotensin II. These results are consistent with inhibitory effects of angiotensin on solute transport by aglomerular tubules.     

Centrally injected angiotensin II trans-synaptically activates angiotensin II-sensitive neurons in the anterior hypothalamic area of rats

Previously, we have demonstrated that pressure-ejected application of angiotensin II onto some neurons in the anterior hypothalamic area (AHA) of rats increases their firing rate. In contrast, pressure application of the angiotensin AT1 receptor antagonist losartan onto AHA neurons blocked the basal firing of the neurons. To investigate possible participation of these AHA neurons in the brain angiotensin system, we examined whether intracerebroventricular injection of angiotensin II results in an activation of angiotensin II-sensitive neurons in the AHA of rats. Intracerebroventricular injection of angiotensin II increased the firing rate of AHA angiotensin II-sensitive neurons. The angiotensin II-induced increase of unit firing in AHA neurons was abolished by pressure application of losartan onto the same neurons. In addition, the angiotensin II-induced increase of firing in AHA neurons was abolished by pressure application of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W7), a calmodulin inhibitor, onto the same neurons. Pressure application of W7 onto AHA neurons affected neither the basal firing rate nor the increase in unit firing induced by pressure application of angiotensin II onto the same neurons. Intracerebroventricular injection of the cholinergic agonist carbachol did not affect the firing rate of angiotensin II-sensitive neurons in the AHA. These findings suggest that intracerebroventricular injection of angiotensin II activates AHA angiotensin II-sensitive neurons via angiotensinergic inputs to the neurons.     

Activation of brain neurons following central hypervolaemia and hypovolaemia: contribution of baroreceptor and non-baroreceptor inputs

In the present study we have used the detection of Fos, the protein product of c-fos, to determine the distribution of neurons in the medulla and hypothalamus that are activated by changes in central blood volume. Experiments were conducted in both barointact and barodenervated conscious rabbits, to determine the contribution of arterial baroreceptors to the pattern of Fos expression evoked by changes in central blood volume, induced either by intravenous infusion of an isotonic modified gelatin solution, or by partial occlusion of the vena cava. These procedures resulted in a significant increase and decrease, respectively, in right atrial pressure over a 60 min period. In control experiments, barointact and barodenervated rabbits were subjected to the identical procedures except that no changes in central blood volume were induced. In comparison with the control observations, central hypervolaemia produced a significant increase in the number of Fos-immunoreactive neurons in the nucleus tractus solitarius, area postrema, the caudal, intermediate and rostral parts of the ventrolateral medulla, supraoptic nucleus, paraventricular nucleus, arcuate nucleus, suprachiasmatic nucleus and median preoptic nucleus. The overall pattern of Fos expression induced by central hypervolaemia did not differ significantly between barointact and barodenervated animals. Similarly, the overall pattern of Fos expression induced by central hypovolaemia did not differ significantly between barointact and barodenervated animals, but did differ significantly from that produced by hypervolaemia. In particular, central hypovolaemia produced a significant increase in Fos expression in the same regions as above, but also in the subfornical organ and organum vasculosum lamina terminalis. In addition, compared with central hypervolaemia, hypovolaemia produced a significantly greater degree of Fos expression in the rostral ventrolateral medulla and supraoptic nucleus. Furthermore, double-labelling for tyrosine hydroxylase immunoreactivity demonstrated that neurons in the ventrolateral medulla that expressed Fos following hypovolaemia were predominantly catecholamine cells, whereas following hypervolaemia they were predominantly non-catecholamine cells. Finally, double-labelling for vasopressin immunoreactivity demonstrated that the number of Fos/vasopressin immunoreactive cells in the supraoptic nucleus was approximately 10 times greater following hypovolaemia compared with hypervolaemia, but there were very few such double-labelled neurons in the paraventricular nucleus in response to either stimulus.The results demonstrate that central hypervolaemia and hypovolaemia each induces reproducible and specific patterns of Fos expression in the medulla and hypothalamus. The degree and pattern of Fos expression was unaffected by arterial baroreceptor denervation, indicating that it is primarily a consequence of inputs from cardiac receptors, together with an increase in the level of circulating hormones such as atrial natriuretic peptide, angiotensin II or vasopressin. Furthermore, the pattern of Fos expression produced by central hypervolaemia and hypovolaemia is distinctly different from that evoked by hypertension and hypotension, respectively [Li and Dampney (1994) Neuroscience 61, 613–634], particularly in hypothalamic regions. These findings therefore indicate that the central pathways activated by changes in blood volume are, at least in part, separate from those activated by changes in arterial pressure.     

Interactions of temperature and angiotensin II in paraventricular neurons of rats in vitro

We recorded extracellular impulse activity of hypothalamic paraventricular neurons ( n=75) in rat brain slices during application of angiotensin II (ANG II, 10(-9)-10(-6) M) and/or temperature changes (32-42 degrees C). ANG II, with a threshold concentration of 10(-8) M, increased the firing rate in more than 80% of the neurons with strongest excitations occurring in bursting neurons. Increasing the temperature also raised the discharge rate in the majority of the neurons, often together with enhanced burst discharges. When ANG II was applied during ongoing sinusoidal temperature changes, its effects were more pronounced at elevated temperatures. These electrophysiological data illustrate that stimulus-encoding properties at the neuronal level can contribute to the interactions between osmoregulatory and thermoregulatory mechanisms including mutual sensitization when different stimuli (here: ANG II and temperature changes) are applied simultaneously.