Effect of chronic administration of the converting enzyme inhibitor enalapril (MK 421) on brain atrial natriuretic peptide receptors in Wistar-Kyoto and spontaneously hypertensive rats

Spontaneously hypertensive rats (SHR) showed lower brain ANP binding density when compared with normotensive Wistar-Kyoto (WKY) rats. In the WKY, angiotensin converting enzyme inhibitor, enalapril (25 mg/kg, p.o. for 14 days), decreased the number of ANP binding sites selectively in the subfornical organ and area postrema. Conversely, enalapril increased ANP binding density in the SHR, but only in the area postrema. Enalapril has central effects on ANP binding sites, specific to the circumventricular organs.     

Increased β2-adrenoceptors in the superior cervical ganglia of genetically hypertensive rats

[¹²⁵I]Iodocyanopindolol binding sites were characterized by autoradiography in the superior cervical ganglia of Wistar-Kyoto (WKY) rats. A high concentration of (−)-[¹²⁵I]iodocyanopindolol binding sites, characterized as β-adrenoceptors by (−)-propranolol displacement, was distributed throughout the ganglia and in the postganglionic (internal carotid) nerve. ICI 118,551, a β₂-selective antagonist, displaced more than 85% of the binding sites, whereas CGP 20712A, a β₁-selective antagonist, displaced less than 10% of the binding sites, indicating that the β-adrenoceptors were primarily of the β₂-subtype. Emulsion autoradiography demonstrated that at least part of the binding sites were associated with principal ganglion cells. Unilateral deafferentation did not modify the number of binding sites in the superior cervical ganglia of WKY or spontaneously hypertensive rat (SHR). These results suggest that at least part of these receptors may correspond to prejunctional β₂-adrenoceptors originated in principal ganglion cells. The concentration of β₂-receptors was increased in the superior cervical ganglia of young and adult SHR when compared to age-matched WKY rats (49% and 39%, respectively). There were no differences in β₂-adrenoceptor number in the stellate ganglia of young and adult WKY and SHR. These results suggest that β₂-adrenoceptor stimulation may be selectively enhanced in some peripheral sympathetic ganglia in SHR and this could play a role in the development and maintenance of the increased symphatetic activity in this strain.     

Autoradiographic localization of binding sites for I-substance P on neurones from cultured rat superior cervical ganglion

Using an autoradiographic receptor binding technique, the distribution of substance P (SP) receptors on cells cultured from the superior cervical ganglia (SCG) of newborn rats was investigated. Binding sites for 125I-Bolton-Hunter-SP were observed on a subpopulation of 35-50% of the ganglion neurones. The percentage of labelled neurones remained constant whether the cultures were seeded densely or sparsely. Variation in the density of labelling was observed on different neuronal clusters. Neuronal cell bodies were often densely labelled, but neuronal processes were rarely labelled. In contrast with the neuronal cells, specific labelling was not associated with other cell types found in this culture preparation, including fibroblasts, glial cells and other non-neuronal supporting cells. These results are interpreted to suggest that there is a subpopulation of SCG neurones which, by virtue of their expressing SP receptors, are responsive to SP and have a physiological role within the ganglion.     

Autoradiographic comparison of muscarinic M1 and M2 binding sites in the CNS of spontaneously hypertensive and normotensive rats

Spontaneously hypertensive rats (SHR) respond with exaggerated pressor responses of central origin in response to pharmacologic stimulation of brain muscarinic receptors when compared with those to normotensive Wistar Kyoto (WKY) rats. At least part of the enhanced response to central muscarinic stimulation may be due to alterations in the expression of one or more of the five subtypes of muscarinic receptors. SHR are also known to exhibit regional alterations in the levels of mRNA encoding the M1, M2 and M4 receptors. In this study, we estimated the number of specific muscarinic receptor binding sites in 12-week-old SHR and WKY by measuring the binding of M1- and M2-selective ligands. Using standard autoradiographic techniques, coronal sections obtained from 12-week-old SHR and WKY were incubated with [³H]pirenzepine or [³H]AFDX 384 to label M1 and M2 receptors, respectively. Although both strains exhibited similar distribution patterns for both binding sites, sections derived from SHR expressed a significant increase in the number of [³H]pirenzepine binding sites compared to normotensive WKY in caudate putamen, CA3 region of the hippocampus, cingulate cortex, substantia nigra, posterior hypothalamic area and tuberomammillary nucleus. An increased number of [³H]AFDX 384 binding sites in SHR were observed in the olfactory tubercle, nucleus accumbens, basolateral amygdaloid nucleus, rostroventrolateral medulla and nucleus paragigantocellularis. Decreases in the number of [³H]AFDX 384 binding sites in SHR were also observed in the parietal cortex, medial geniculate, and lateral hypothalamic area. Statistically significant site-selective differences in binding densities between strains ranged from 4.0% to 35.5% of WKY means. These alterations in the expression of M1 and M2 binding sites in cardiovascular regions may contribute to the strain's hyper-responsiveness to cholinergic drugs and possibly to the appearance of other autonomic or behavioral phenotypes exhibited by SHR, including the hypertensive state itself.     

Angiotensin II binding sites in the hamster brain: localization and subtype distribution

This study was designed to characterize the distribution of angiotensin II (AII) binding sites in the hamster brain. Brain sections were incubated with [¹²⁵I][sar¹, ile⁸]-angiotensin II in the absence and presence of angiotensin II receptor subtype selective compounds, losartan (AAT, subtype) and PD123177 (AT₂ subtype). Binding was quantified by densoritometric autoradiograms and localized by comparison with adjacent thionin stained sections. The distribution of AII binding sites was similar to that found in the rat, with some exceptions. [¹²⁵I][sar¹, ile⁸]-angiotensin II binding was not evident in the subthalamic nucleus and thalamic regions, inferior olive, suprachiasmatic nucleus, and piriform cortex of the hamster, regions of prominent binding in the rat brain. However, intense binding was observed in the interpeduncular nucleus and the medial habenula of the hamster, nuclei void of binding in the rat brain. Competition with receptor subtype selective compounds revealed a similar AII receptor subtype profile in brain regions where binding is evident in both species. One notable exception is the medial geniculate nucleus, predominately AT₁ binding sites in the hamster but AT₂ in the rat. Generally, the AII binding site distribution in the hamster brain parallels that of the other species studied, particularly in brain regions associated with cardiovascular and dipsogenic functions. Functional correlates for AII binding sites have not been elucidated in the majority of brain regions and species mismatches might provide clues in this regard.     

Fine structure of the cells containing large granular vesicles in the superior cervical ganglia of rat embryos

An electron microscopic study has been performed on the superior cervical ganglia of 11- to 21-day-old rat embryos after fixation with glutaraldehyde and osmium tetroxide. The ganglia of 11-day-old and older embryos are known to consist of cells which show catecholamine fluorescence, the intensity of which varies from weak to bright. Electron microscopically, the ganglia of 11- and 12-day-old embryos were observed to consist of a dense group of cells among the loose mesenchyme. The cells showed a great variation in nuclear morphology and only rarely contained granular vesicles. Only few cells represented the undifferentiated, primitive sympathetic cell type described in previous studies. Therefore, in the rat superior cervical ganglion the ultrastuctural differentiation may be initiated even before the cells complete their migration.From embryonic day 14, three types of cell containing varying numbers of large granular vesicles were distinguished on the basis of nuclear morphology. Those type 1 cells that contained numerous granular vesicles closely resemble the postnatal SIF cells. Those type 2 cells that possessed a great number of granular vesicles may represent an intermediate form, namely the large intensely fluorescent cell described in prenatal ganglia but only exceptionally found in early postnatal ganglia. The appearance of the type 2 cells with only a few granular vesicles is identical with that of the sympathetic cells representing the early stages of maturation. The type 3 cells correspond with the late maturing principal neuron. These cells were never seen to contain more than a few large granular vesicles.     

Increased density of substance P binding sites in specific brainstem nuclei of spontaneously hypertensive rats

Substance P binding sites were quantitated in 16-week-old spontaneously hypertensive rats (SHR) and age-matched normotensive Wistar-Kyoto rats by incubation of brain sections with¹²⁵I-Bolton-Hunter substance P, autodiography and microdensitometry. The maximum binding capacity (B_max) was significantly increased in the dorsal motor nucleus of the vagus, in the hypoglossal, ambiguus and inferior olivary nuclei, and in lobes 9 and 10 of the vermis cerebelli of SHR.     

Specific angiotensin II binding sites in the rat stellate and superior cervical ganglia

Angiotensin II binding sites were localized and quantified in rat stellate and superior cervical ganglia by quantitative autoradiography using 125I-Sar1-angiotensin II as a ligand. Both ganglia possess a single class of angiotensin II binding sites. High concentrations of binding sites were localized in the areas rich in principal ganglion cells. These binding sites might mediate the facilitatory action of angiotensin II on the ganglionic transmission.     

Pituitary adenylate cyclase-activating peptide (PACAP) and PACAP type 1 receptor expression in regenerating adult mouse and rat superior cervical ganglia in vitro

Pituitary adenylate cyclase-activating polypeptide (PACAP), a regulatory peptide belonging to the vasoactive intestinal peptide (VIP) family, is widely distributed in the central and peripheral nervous system. Recent studies have shown that PACAP expression is upregulated in sensory neurons in response to axonal injury. Here we report that PACAP and PACAP type 1 receptors are located in rat and mouse superior cervical ganglia (SCG). PACAP-immunoreactivity (-IR) was demonstrated in preganglionic fibers, whereas only occasional PACAP-IR cell bodies could be observed. In situ hybridization histochemistry using ³⁵S-labeled deoxyribonucleotide probes confirmed that PACAP mRNA was present only in occasional cell bodies. In contrast, PACAP type 1 receptor mRNA was expressed in virtually all cell bodies within the ganglia. After removal and culturing of the SCG for 24 h, there was a marked increase in PACAP mRNA, whilst PACAP type 1 receptor mRNA expression appeared to be downregulated in most nerve cell bodies except for a few scattered neurons displaying a strong upregulation. The total specific binding of PACAP to isolated SCG membranes as assayed by [¹²⁵I]PACAP-27 binding showed an increase in SCG cultured for 48 h. PACAP-27 neither affected axonal outgrowth from the cultured SCG nor the survival of cells within the SCG. We conclude that PACAP and PACAP receptors are rapidly upregulated in sympathetic ganglia in response to axonal injury and that PACAP may play a role during nerve regeneration.     

Role of neuron soma firing in the restoration of neurotensin store in sympathetic preganglionic neuron terminals after stimulus-evoked depletion

We have previously shown that prolonged preganglionic stimulation (e.g., 40 Hz 20 min) depletes the neurotensin (NT) store of preganglionic axon terminals in the stellate ganglion (SG) of the cat and that replenishment of the store requires several days. The present study investigates the mechanisms which control turnover of the NT store in sympathetic preganglionic axon terminals. NT content of the SG and of the preganglionic axons which innervate it was determined by radioimmunoassay in the anesthetized cat. This study shows that, during the 24 h after 40-Hz 20-min stimulation of the preganglionic input to the SG, the rate of NT accumulation proximal to a ligature on the stimulated input is three times that observed in the control. The poststimulus increase in NT accumulation rate is prevented by treatment with protein-synthesis inhibitors. When the centripetal propagation of action potentials from the stimulus site on the preganglionic axons is prevented by a tetrodotoxin bloxk applied locally during the stimulation period, the poststimulus increase in NT accumulation rate and the replenishment of the store are both prevented. These data suggest that the level of activity of the neuron regulates NT supply to the axon terminals, presumably by regulating NT synthesis. Thus, in the sympathetic preganglionic neuron, the action potential provides a mechanism for matching peptide synthesis to release.     

In vitro and in vivo evidence of neurotensin release from preganglionic axon terminals in the stellate ganglion of the cat

We have previously shown that the neurotensin (NT) store in preganglionic axon terminals of the cat stellate ganglion (SG) is reversibly depleted by prolonged preganglionic stimulation. The present study addresses the questions of whether the preganglionic axon terminals release NT in response to depolarizing stimuli in vitro and whether in vivo NT is released by the tonic firing of the sympathetic preganglionic neurons. Slices of the SG of the anaesthetized cat, maintained in oxygenated Ringer solution, released NT. The efflux increased when the K concentration was increased from 5 to 25 or 45 mM or when veratridine was added to the medium. In Ca-free medium, efflux was suppressed. The effect of veratridine was blocked by tetrodotoxin (TTX). In awake, freely moving cats, in which TTX was applied for 4 days to the preganglionic input of the right SG, the NT content of this ganglion doubled by comparison with the left SG. Since NT accumulates proximal to a ligature on the preganglionic input of the SG, the increased NT content is likely to result from suppression of action potential-dependent release while influx into the terminals persists. This result suggests that the steady state of the NT store in sympathetic preganglionic terminals is the result of a steady influx from the soma balanced by action potential-dependent loss, presumably release.     

Naloxone-sensitive inhibition of nicotinic transmission in the superior cervical ganglion of the cat

In anesthetized, artificially ventilated cats, pretreated with the muscarinic antagonist scopolamine, the effect of naloxone on the efficacy of nicotinic transmission in the superior cervical ganglion was used as a test of endogenous opioid release by the preganglionic axons. The cervical sympathetic trunk (CST) was split into two bundles. The compound action potential (CAP), evoked by supramaximal low-frequency stimulation (0.25 Hz) of one CST bundle, was recorded from a postganglionic nerve of the superior cervical ganglion. Partial block with hexamethonium was used to reduce the 'safety factor' of nicotinic transmission. A conditioning train (5 Hz, 40 s, supramaximal) to the other CST bundle (heterosynaptic conditioning) inhibited the CAP. At the peak of the inhibition the CAP was attenuated by 51 +/- 4%. Recovery was 90% complete in 201 +/- 28 s. The inhibition was antagonized in a dose-dependent manner by i.v. naloxone with an apparent IC50 of 60 +/- 12 micrograms/kg. The maximum effect obtained with naloxone was an 80% decrease in the magnitude of the inhibition. Magnitude and duration of the heterosynaptic inhibition were related to frequency, duration and intensity of the conditioning train. Naloxone-sensitive inhibition was observed with frequencies of the conditioning train as low as 0.5 Hz. A train (5 Hz, 40 s) to the postganglionic nerves produced a naloxone-insensitive depression of the orthodromic test CAP which was of smaller amplitude and duration than when the conditioning train was applied to the preganglionic axons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neurokinin-1 receptors and spinal cord control of blood pressure in spontaneously hypertensive rats

In this study we examined blood pressure and heart rate responses to intrathecal administration of a synthetic NK1-receptor agonist, H₂N–(CH₂)₄–CO–Phe–Phe–Pro–NmeLeu–Met–NH2 (GR 73,632), in spontaneously hypertensive rats (SHR) and their progenitor strain, the Wistar–Kyoto rat (WKY). Sodium pentobarbitone anaesthetised rats with implanted intrathecal catheters were paralysed (pancuronium dibromide) and artificially ventilated. Injection of GR 73,632 at the T9 spinal level evoked dose-dependent increases in mean arterial pressure (MAP) in WKY and SHR. SHR had a lower MAP response threshold than WKY but increase in response with increasing dose was less in SHR than WKY. Biphasic blood pressure responses at high doses were observed in both strains. Prior administration of the NK1-receptor antagonist (3aR,7aR)-7,7-diphenyl-2-[1-imino-2(methoxyphenyl)ethyl] perhydroisoindol-4-one (RP 67,580) significantly reduced the pressor response in WKY but not SHR. The depressor response was not attenuated in either strain.     

A comparative autoradiographic study of the distributions of substance P and eledoisin binding sites in rat brain

The relative potencies of tachykinin peptide analogs competing for binding of [125I]Bolton Hunter-conjugated substance P ([125I]BH-SP) or [125I]Bolton Hunter-conjugated eledoisin ([125I]BH-ED) in slide-mounted rat brain sections are very different, indicating the presence of two distinct tachykinin binding sites. The structure-activity profiles resemble those described in peripheral bioassay studies in which two tachykinin receptors have been postulated. Autoradiography of the two iodinated ligands bound with selective and one-site in vitro incubation conditions shows two discrete and distinctly different distribution patterns in brain. Binding sites for [125I]BH-ED are densely distributed in the accessory olfactory bulb, intermediate layers of the cerebral neocortex, portions of the hippocampal CA fields, hypothalamic supraoptic and paraventricular nuclei, central portions of the interpeduncular nucleus, sphenoid nucleus, medial subdivision of the solitary tract complex, and the substantia gelatinosa of the spinal cord. Binding sites for [125I]BH-SP are present in many of these same structures, but the densities and distribution patterns are different. In addition, [125I]BH-SP binds in numerous structures not labeled by [125I]BH-ED. Neither pattern matches the locations of terminations of endogenous tachykinin pathways marked by immunohistochemistry. The results suggest that it would be inappropriate to name brain tachykinin receptors according to the endogenous ligand which binds with highest affinity.     

The site of inhibitory action of endogenous opioids in the superior cervical ganglion of the cat

A low-frequency stimulus train to the preganglionic input inhibits synaptic transmission in the superior cervical ganglion (SCG) of the cat. The inhibition is blocked by naloxone as well as by selective antagonists at mu and delta opiate receptors, which suggests that the mediator is an endogenous opioid [27, 29]. Exogenous opioid peptides, including methionine-enkephalin (Met-Enk), which is present in preganglionic axons of the SCG, inhibit ganglionic transmission by a naloxone-sensitive mechanism. In the present study we test, in the anesthetized cat, whether the naloxone-sensitive synaptic inhibition is mediated by a pre- and/or post-synaptic mechanism. As a test of presynaptic inhibition, we measured the acetylcholine (ACh) released by preganglionic stimulation into the venous effluent of the perfused SCG. As a test of post-synaptic inhibition, we measured the effect of a preganglionic conditioning train on the ganglion cell firing evoked by ganglion-stimulant drugs injected into the arterial supply of the ganglion. In presence of naloxone (3 μM), which blocked the synaptic inhibition, the amount of ACh released by stimulated preganglionic axons did not change. Thus, the endogenous opioid which mediates the naloxone-sensitive inhibition does not act by depressing ACh release. In contrast, the ganglion cell firing evoked by ganglion-stimulant drugs was markedly depressed by a conditioning train, and naloxone blocked the depression, which suggests that the endogenous mediator of the naloxone-sensitive inhibition acts postsynaptically to decrease the excitability of ganglion cells. Exogenous Met-Enk depressed both ACh release by preganglionic stimulation and the firing of ganglion cells evoked by ganglion-stimulant drugs. These findings suggest that although opiate receptors in the cat SCG are present at both pre- and post-synaptic sites, under the conditions of the present experiment the naloxone-sensitive, synaptically mediated inhibition is mediated exclusively by activation of post-synaptic opiate receptors.     

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 alpha-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.     

Role of prostaglandin,endothelin and sympathetic nervous system on the L-NAME-induced pressor responses in spontaneously hypertensive rats

We tested the hypothesis that in spontaneously hypertensive rat (SHR) NO produced centrally influences the resting arterial blood pressure by attenuating mechanisms involving prostaglandins, angiotensin II, endothelin and sympathetic nervous system. L-NAME (200 micro g/5 micro l), an inhibitor of NO synthase, administered intracerebroventricularly (i.c.v.) to awake and freely moving rats increased mean arterial blood pressure (MABP) in a biphasic pattern: an early transient increase within 1 min and a late prolonged response starting at 45 min and persisting for the duration of experiment (180 min). The two pressor responses involve different neurochemical mechanisms and, based on their latencies, they appear to reflect different anatomical sites of action of L-NAME. The late, but not the early pressor response, was prevented by pretreatment with chlorisondamine (2.5 mg/kg, i.v.), a ganglionic blocker, indicating its dependence on the sympathetic nervous system. Both pressor responses were abolished by i.c.v. pretreatment with indomethacin (200 micro g/5 micro l, i.c.v.), an inhibitor of cyclo-oxygenase, showing that they are mediated by prostaglandin(s). In contrast, losartan (25 micro g/5 micro l), an angiotensin II AT(1) receptor antagonist, had no effect. The initial pressor response was also attenuated by pretreatment with the endothelin ET(A)/ET(B) receptor antagonist, PD 145065 (48 micro g/2 micro l, i.c.v.). Intravenous pretreatment with another ET(A)/ET(B) receptor antagonist, L-754,142 (15 mg/kg as a bolus+15 mg/kg/h for 180 min), however, attenuated both responses to L-NAME. It is possible that L-754,142 crossed the blood-brain barrier and blocked, in addition, central ET(A)/ET(B) receptors. These studies show that NO synthesized in the brain attenuates pressor mechanisms involving prostaglandin, endothelin and sympathetic nervous system, but not angiotensin II, to modulate resting arterial blood pressure.     

Norepinephrine turnover in pineal gland and superior cervical ganglia. Changes after gonadotrophin administration to castrated rats

Summary The effects of FSH and LH on norepinephrine (NE) turnover in pineal gland and superior cervical ganglia of castrated female rats were examined by measuring the decline of [³H]NE concentration in the organ 30–120 min after a single i.v. injection of the labeled amine. Both FSH and LH treatment significantly decreased [³H]NE turnover in the pineal gland whereas only FSH accelerated it in the ganglia. As compared to controls, the initial uptake of [³H]NE was lower in the pineal gland of FSH- and LH-treated animals, and higher in the superior cervical ganglia of rats injected with FSH. Neither FSH nor LH modified [³H]NE turnover in heart or adrenal gland, nor changed their endogenous catecholamine content. Pineal monoamine oxidase (MAO) activity type B (assaying by using-phenylethylamine as substrate) was decreased by FSH or LH injection. Only FSH modified MAO activity in the ganglia by increasing significantly type A enzyme (assaying by using serotonin as substrate). These results suggest that FSH and LH affect significantly NE metabolism in pineal gland and superior cervical ganglia of spayed rats.     

Sympathetic preganglionic neurons contain nitric oxide synthase and project to the superior cervical ganglion: Combined application of retrograde neuronal tracer and NADPH-diaphorase histochemistry

Nitric Oxide (NO), which was initally identified as an endothelium-derived relaxing factor, has recently been demonstrated to be a neuronal messenger in central and peripheral nervous systems. In the present study, we examined the possibility of NO producing neurons in the intermediolateral (IML) cell collum of the thoracic spinal cord (Th) project to the superior cervical ganglion (SCG). First, we observed the NADPH-diaphorase-positive/nitric oxide synthase (NOS)-immunoreactive neurons of the IML and the dorsal part of the central canal at the level of Th1–Th3, and numerous fiber-stainings in the superior cervical ganglion. Second, after injecting WGA-HRP (wheat germ agglutinin-horse radish peroxidase complex), a retrograde neuronal tracer, into the SCG, and developing WGA-immunohistochemistry and the NADPH-diaphorase histochemistry in the same sections, we detected double-labeled neurons in the IML. These findings provide evidence that sympathetic preganglionic NO producing neurons directly innervate to the SCG.