Altered L-DOPA systems for blood pressure regulation in the lower brainstem of spontaneously hypertensive rats

Recent findings have enhanced our understanding of the roles played by the L-DOPA system in the baroreceptor reflex and in blood pressure regulation in the lower brainstem. L-DOPA is probably a neurotransmitter of primary baroreceptor afferents terminating in depressor sites of the nucleus tractus solitarii (NTS). It also seems to be a neurotransmitter in depressor sites of the caudal ventrolateral medulla (CVLM) and in pressor sites of the rostral ventrolateral medulla (RVLM) of normotensive Wistar rats. We have explored whether or not presynaptic and postsynaptic functions of the L-DOPA system in these areas are altered to maintain hypertension in adult spontaneously hypertensive rats, as compared with age-matched Wistar Kyoto rats. In this review article, we survey the roles of the L-DOPA system in the baroreceptor reflex and in blood pressure regulation in the rat lower brainstem.     

A comparison of intravascular pressure monitoring system contamination and patient bacteremia with use of 48- and 72-hour system change intervals

We hypothesized that there may be a significant difference in the neuronal composition of the baroreceptor reflex pathway between normotensive Wistar Kyoto (WKY) and spontaneously hypertensive SHR rats. Using the double-immunoreactive (IR) method, the topology and numbers of barosensitive neurons that contain glutamate (Glu), glutamic acid decarboxylase (GAD), tyrosine hydroxylase (TH), phenylethanolamine N-methyltransferase (PNMT) and choline acetyltransferase (ChAT) were compared between the two strains. The control rats were sham-operated only for cannulation of the trachea and femoral artery/vein. The test rats were injected with the pressor agent phenylephrine to raise blood pressure and stimulate arterial baroreceptors. In both the control and test experiments, the c-Fos/Glu-, GAD-, TH- and PNMT-IR neurons were found in the nucleus tractus solitarii (NTS) and ventrolateral medulla (VLM), while the FosB/ChAT-IR neurons were found in the NTS, dorsal motor nucleus of the vagus (DMX) and nucleus ambiguus (AMB). In the control experiment, no significant difference in numbers was recognized in any of the double-IR neurons between the two strains. In the test experiment, the numbers of FosB/ChAT-IR neurons in the NTS, DMX and AMB were significantly smaller in SHR than in WKY. The numbers of c-Fos/TH-IR neurons in the caudal VLM were significantly larger in SHR than in WKY. These results suggest that a smaller number of barosensitive cholinergic neurons in the DMX and AMB in SHR causes the weaker baroreceptor-cardiac vagal reflex in SHR, and that a larger number of barosensitive catecholaminergic neurons in the caudal VLM in SHR are involved in the stronger baroreceptor-vasopressin reflex in SHR.     

c-Fos expression in brain in response to hypotension and hypertension in conscious rats

Hypotension- and hypertension-evoked expression of the protein product, Fos, of the immediate early gene c-fos was assessed throughout the rat brain as an approach for describing the neuronal populations that respond to alterations in arterial blood pressure. Conscious, chronically catheterized rats were treated with the vasoconstricting drug phenylephrine or the vasodilatating drug hydralazine to increase or decrease, respectively, arterial pressure by approx. 40 mm Hg for 90 min. Rats were then anesthetized, fixed by vascular perfusion, and sections representing the entire brain were processed for the immunocytochemical localization of Fos. In control rats treated with isotonic saline, few Fos-positive neurons were observed. In contrast, phenylephrine and hydralazine treatments resulted in different, yet reproducible, patterns of Fos expression in the brain, with hydralazine evoking Fos expression in more brain regions than phenylephrine. Brain regions containing Fos-positive neurons in rats treated with hydralazine included nucleus tractus solitarius, area postrema, caudal ventrolateral medulla, rostral ventrolateral medulla, bed nucleus of the stria terminalis, amygdala, paraventricular nucleus, supraoptic nucleus, subfornical organ and the Islands of Calleja. The nucleus tractus solitarius, paraventricular nucleus and the amygdala also contained Fos-positive neurons in phenylephrine-treated rats, although the number of Fos-positive neurons was always less than that noted in the hydralazine-treated rats and the location of Fos-positive neurons within these regions tended to differ between treatments. These results generally fit within an emerging understanding of brain circuitry underlying cardiovascular regulation.     

Intracerebroventricular CRF inhibits cold restraint-induced c-fos expression in the dorsal motor nucleus of the vagus and gastric erosions in rats

Acute exposure to cold-restraint induces vagal-dependent gastric erosions associated with activation of neurons in the dorsal motor nucleus of the vagus (DMN) in rats. The influence of intracerebroventricular (i.c.v.) injection of corticotropin-releasing factor (CRF) (10 micrograms) on c-fos expression in the brain and gastric erosions induced by 3 h cold-restraint was investigated in conscious rats. In cold-restraint exposed rats, CRF injected i.c.v. inhibited gastric erosions and the number of Fos positive neurons in the DMN by 93 and 72%, respectively, while Fos labelling in the nucleus tractus solitarius (NTS) was increased by 5-fold compared with vehicle group. c-fos expression was also induced in the central amygdala by i.c.v. CRF, unlike the vehicle-injected group exposed to cold-restraint. c-fos expression induced by cold-restraint in the raphe pallidus (Rpa) and paraventricular nucleus of the hypothalamus was not altered by i.c.v. CRF. These data indicate that central CRF-induced gastric protection results from the inhibition of DMN neuronal activity enhanced by cold-restraint. CRF action on DMN neurons may be related to the increase in the NTS and central amygdala inputs leading to inhibition of DMN neurons rather than to the decrease in the excitatory input from the caudal raphe projections to the DMN.     

Effect of bilateral lesions of the solitary tract nucleus on arterial blood pressure in spontaneously hypertensive rats of Okamoto strain

Bilateral electrolytic lesions of the solitary tract nucleus in control Wistar albino rats as well as in Okamoto rats with spontaneous arterial hypertension caused a considerable rise in the arterial blood pressure. Quantitative comparison of blood pressure rise in normotensive and hypertensive rats demonstrated that the rise in the systolic and mean blood pressure was not significantly different in both groups of animals. However, the rats with spontaneous hypertension reacted, with a greater rise in the diastolic pressure. A characteristic feature observed in the normotensive rats was a high rise in pulse pressure following lesion of the solitary tract nucleus while in the rats with spontaneous hypertension this change was not found. The authors conclude that increased peripheral vascular resistance in the rats with spontaneous hypertension is not due to inhibition or resetting of the baroreceptor reflex.     

Psychoneurotic characteristics of patients with anorexia nervosa before and after treatment and at follow-up 4-7 years later

In normotensive (NR) and spontaneously hypertensive rats (SHR) the cardiac vagal component of baroreceptor reflex was checked before and after intravenous infusion of clonidine (25-30 microgram/kg). 1) The decrease of heart rate due to the arterial baroreceptor reflex was the same in NR and in SHR. 2) Intravenous injection of clonidine produced much greater decrease of the heart rate in SHR than in NR. 3) After administration of clonidine the stimulation of arterial baroreceptors decreased the heart rate to the same low level in NR and SHR. In these conditions the maximal neurogenic slowing of the heart rate was probably achieved. 4) Atropine abolished almost completly heart rate change following baroreceptor stimulation or/and clonidine administration.     

Role of endogenous carbon monoxide in central regulation of arterial pressure

We investigated the contribution of neural mechanisms to the arterial pressure increase produced by zinc deuteroporphyrin 2,4-bis glycol (ZnDPBG), an inhibitor of endogenous carbon monoxide synthesis. The arterial baroreceptor reflex control of heart rate was examined in rats with and without ZnDPBG pretreatment (45 micromol/kg IP) by analysis of the arterial pressure-heart rate relationship during infusions of phenylephrine or sodium nitroprusside to vary arterial pressure. ZnDPBG increased arterial pressure from 110 +/- 3 to 126 +/- 2 mm Hg without eliciting bradycardia. The maximum gain of the heart rate response to changes in arterial pressure was attenuated by ZnDPBG treatment (-1.9 +/- 0.3 versus -4.8 +/- 1.0 bpm/mm Hg). The possibility that ZnDPBG elevates arterial pressure by attenuating baroreceptor reflex function was addressed by comparing the pressor response to ZnDPBG (45 micromol/kg IP) in rats with and without sinoaortic denervation. The pressor effect of ZnDPBG was similar in rats with and without arterial baroreceptor deafferentation, implying that the increase in pressure is not simply the consequence of attenuated baroreceptor reflex function per se. The possibility that ZnDPBG increases arterial pressure via an effect on the nucleus tractus solitarii (NTS) also was investigated. ZnDPBG (1 nmol in 100 nL) injected into the NTS of rats increased arterial pressure from 111 +/- 4 to 126 +/- 5 mm Hg, and this effect was reversed by an ipsilateral microinjection of carbon monoxide into the NTS. Accordingly, the pressor effect of ZnDPBG may rely on inhibition of carbon monoxide production in the NTS. This implies that carbon monoxide formed by brain heme oxygenase plays a role in the central regulation of arterial pressure.     

Effects of sinoaortic denervation on Fos expression in the brain evoked by hypertension and hypotension in conscious rabbits

We have previously shown [Li and Dampney (1994) Neuroscience 61, 613-634] that periods of sustained hypertension and hypotension each induces a distinctive and reproducible pattern of neuronal expression of Fos (a marker of neuronal activation) in specific regions of the brainstem and forebrain of conscious rabbits. The aim of this study was to determine the contribution of afferent inputs from arterial baroreceptors to the activation of neurons in these various brain regions that is caused by a sustained change in arterial pressure. Experiments were carried out on rabbits in which the carotid sinus and aortic depressor nerves were cut in a preliminary operation. Following a recovery period of seven to 10 days, a moderate hypertension or hypotension (increase or decrease in arterial pressure of 20-30 mmHg) was induced in conscious barodenervated rabbits for 60 min by the continuous infusion of phenylephrine or sodium nitroprusside, respectively. In control experiments, barodenervated rabbits were subjected to the identical procedures except that they were infused with the vehicle solution alone. Compared with the effects seen in barointact rabbits, [Li and Dampney (1994) Neuroscience 61, 613-634] the number of neurons that expressed Fos in response to hypertension was reduced by approximately 90% in the nucleus of the solitary tract and in the caudal and intermediate parts of the ventrolateral medulla. In supramedullary regions, baroreceptor denervation resulted in a reduction of approximately 60% in hypertension-induced Fos expression in the central nucleus of the amygdala and in the bed nucleus of the stria terminalis, but no significant reduction in the parabrachial complex in the pons. Following hypotension, the number of neurons that expressed Fos in barodenervated rabbits, compared with barointact rabbits, [Li and Dampney (1994) Neuroscience 61, 613-634] was reduced by approximately 90% in the nucleus of the solitary tract, area postrema, and caudal, intermediate and rostral parts of the ventrolateral medulla. Baroreceptor denervation also resulted in a similar large reduction in hypotension-induced Fos expression in many supramedullary regions (locus coeruleus, midbrain periaqueductal grey, hypothalamic paraventricular nucleus, and in the central nucleus of the amygdala and the bed nucleus of the stria terminalis in the basal forebrain). In the supraoptic nucleus, hypotension-induced Fos expression in barodenervated rabbits was reduced by 75% compared to barointact animals, but was still significantly greater than in control animals. There was also a high level of Fos expression, much greater than in control animals, in the circumventricular organs surrounding the third ventricle (subfornical organ and organum vasculosum lamina terminalis). The results indicate that in conscious rabbits the activation of neurons that occurs in several discrete regions at all levels of the brain following a sustained change in arterial pressure is largely dependent upon inputs from arterial baroreceptors, with the exception of neurons in the circumventricular organs surrounding the third ventricle that are activated by sustained hypotension. The latter group of neurons are known to project to vasopressin-secreting neurons in the supraoptic nucleus, and may therefore via this pathway trigger the hypotension-induced release of vasopressin that occurs in the absence of baroreceptor inputs.     

Suppression of postischemic hippocampal nerve growth factor expression by a c-fos antisense oligodeoxynucleotide

We examined the uptake and distribution of an antisense phosphorothioated oligodeoxynucleotide (s-ODN) to c-fos, rncfosr115, infused into the left cerebral ventricle of male Long-Evans rats and the effect of this s-ODN on subsequent Fos, NGF, neurotrophin-3 (NT-3), and actin expression. To establish the uptake and turnover of s-ODN in the brain, we studied the copurification of the immunoreactivity of biotin with biotinylated s-ODN that was recovered from different regions of the brain. A time-dependent diffusion and the localization of s-ODN were further demonstrated by labeling the 3'-OH terminus of s-ODN in situ with digoxigenin-dUTP using terminal transferase and detection using anti-digoxigenin IgG-FITC. Cellular uptake of the s-ODN was evident in both the hippocampal and cortical regions, consistent with a gradient originating at the ventricular surface. Degradation of the s-ODN was observed beginning 48 hr after delivery. The effectiveness of c-fos antisense s-ODN was demonstrated by its suppression of postischemic Fos expression, which was accompanied by an inhibition of ischemia-induced NGF mRNA expression in the dentate gyrus. Infusion of saline, the sense s-ODN, or a mismatch antisense s-ODN did not suppress Fos expression. That this effect of c-fos antisense s-ODN was specific to NGF was demonstrated by its lack of effect on the postischemic expression of the NT-3 and beta-actin genes. Our results demonstrate that c-fos antisense s-ODN blocks selected downstream events and support the contention that postischemic Fos regulates the subsequent expression of the NGF gene and that Fos expression may have a functional component in neuroregeneration after focal cerebral ischemia-reperfusion.     

High-performance liquid chromatographic analysis of the new antitumour drug N-benzoylstaurosporine (CGP 41 251) and four potential metabolites in micro-volumes of plasma

Molecular events underlying the mechanism by which brain injury elicits delayed transneuronal degeneration of neurons remote from the site of initial injury are not well understood. In rats, acute injury of the caudate nucleus (CN) and globus pallidus (GP) by local injection of excitotoxic ibotenic acid (IA) or by transient forebrain ischemia resulted in delayed cell death of neurons in the substantia nigra reticulata (SNr). To elucidate the involvement of glutamate receptor mediated hyperactivity of neurons produced by loss of inhibitory inputs in this delayed degeneration of SNr neurons, the region-specific expression of an immediate early gene, c-fos, and the effect of glutamate receptor antagonists on the c-fos expression were examined by using immunocytochemical and in situ hybridization analysis. Following unilateral IA-injection into the CN and GP, a robust expression of c-fos mRNA and Fos protein was induced specifically in neurons of both subthalamic nucleus (STN) and SNr deafferented by the IA-lesions 36 h after IA-injection. The delayed expression of Fos-protein in SNr neurons lasted for 48 h longer than that in STN neurons. Following unilateral IA-injection confined to the CN, an intense but short-term expression of Fos-protein was exhibited only in neurons of the deafferented SNr. c-fos mRNA and Fos protein were not expressed in neurons of the substantia nigra compacta at any time points examined. The induction of c-fos mRNA and Fos protein in neurons of the STN and SNr following IA-lesions of the CN and GP was reduced markedly by non-NMDA receptor antagonist (GYKI52466), but not by NMDA receptor antagonist (MK-801). The region-specific c-fos expression implies that deprivation of inhibitory afferents (disinhibition) due to destruction of presynaptic neurons can induce increased activity of postsynaptic neurons. The effect of GYKI52466 on the c-fos gene expression in neurons of the deafferented STN and SNr suggests that activation of non-NMDA receptors may be involved in a pathophysiological cascade for the transneuronal degeneration of SNr neurons.     

Spatially and temporally differentiated patterns of c-fos expression in brainstem catecholaminergic cell groups induced by cardiovascular challenges in the rat

Brainstem catecholaminergic neurons have been implicated as mediating adaptive autonomic and neuroendocrine responses to cardiovascular challenges. To clarify the nature of this involvement, immuno- and hybridization histochemical methods were used to follow c-fos expression in these neurons in response to acute stimuli that differentially affect blood pressure and volume. From low basal levels, hypotensive hemorrhage (15%) provoked a progressive increase in the number and distribution of Fos-immunoreactive (ir) nuclei in the nucleus of the solitary tract (NTS), the A1 and C1 cell groups of the ventrolateral medulla, and in the pontine A5, locus coeruleus, and lateral parabrachial cell groups peaking at 2.0-2.5 hours after the challenge. Fos-ir ventrolateral medullary neurons, subsets of which were identified as projecting to the paraventricular hypothalamic nucleus or spinal cord, were predominantly aminergic, whereas most of those in the NTS were not. Infusion of an angiotensin II antagonist blunted hemorrhage-induced Fos expression in the area postrema, and attenuated that seen elsewhere in the medulla and pons. Nitroprusside-induced isovolemic hypotension yielded a pattern of c-fos induction similar to that seen following hemorrhage, except in the area postrema and the A1 cell group, where the response was muted or lacking. Phenylephrine-induced hypertension stimulated a restricted pattern of c-fos expression, largely limited to induced hypertension stimulated a restricted pattern of c-fos expression, largely limited to non-aminergic neurons, whose distribution in the NTS conformed to the termination patterns of primary baroreceptor afferents, and in the ventrolateral medulla overlapped in part with those of vagal cardiomotor and depressor neurons. These findings underscore the importance of brainstem catecholaminergic neurons in effecting integrated homeostatic responses to cardiovascular challenges and their ability to responding strategically to specific modalities of cardiovascular information. They also foster testable predictions as to effector neuron populations that might be recruited to respond to perturbations in individual circulatory parameters.     

Comparison of the chronic toxicity of piroxantrone, losoxantrone and doxorubicin in spontaneously hypertensive rats

We have determined the time course, the spatial spread in brain tissue, and the intracellular distribution of biotin- and fluorescein-labeled phosphorothioate oligodeoxynucleotides (ODNs) following single injections into the rat striatum or the lateral ventricle. These time and space parameters were correlated with the ability of c-fos phosphorothioate antisense ODNs to suppress the induction of Fos protein by cocaine. A rapid and dose-dependent tissue penetration of labeled ODNs was observed following either intrastriatal or intraventricular injections of a constant sample volume. Inspection of tissue sections by confocal microscopy uncovered a distinct change in the intracellular disposition of labeled ODNs during the 24 h post-injection period. At 1, 6 and 12 h, the vast majority of the fluorescent signal was confined to the interstitial spaces throughout the zone penetrated by ODNs. Neuronal nuclei displayed faint labeling along the outer portion of the nucleus at 1 and 6 h post-injection. At these time-points, ODNs were not detected in the cytoplasm. By 16 h, ODNs were barely detectable in the extracellular space and absent from neuronal nuclei. Instead, ODNs were seen in large cytoplasmic granules of neurons throughout the tissue zone penetrated by the ODNs. Experiments with intrastriatal injections of antisense ODNs to c-fos mRNA revealed Fos suppression between 3 and 12 h, but not at 16 and 24 h. This combined analysis has revealed that (1) restricted tissue penetration by ODNs limits their antisense effects on protein expression, and (2) depletion of extracellular ODNs and sequestration of c-fos antisense ODNs into large intracellular granules coincides with the loss of their biological activity.     

Characterization of a potent and specific class of antisense oligonucleotide inhibitor of human protein kinase C-alpha expression

The use of antisense oligonucleotides to inhibit the expression of targeted mRNA sequences is becoming increasingly commonplace. Although effective, the most widely used oligonucleotide modification (phosphorothioate) has some limitations. In previous studies we have described a 20-mer phosphorothioate oligodeoxynucleotide inhibitor of human protein kinase C-alpha expression. In an effort to identify improved antisense inhibitors of protein kinase C expression, a series of 2' modifications have been incorporated into the protein kinase C-alpha targeting oligonucleotide, and the effects on oligonucleotide biophysical characteristics and pharmacology evaluated. The incorporation of 2'-O-(2-methoxy)ethyl chemistry resulted in a number of significant improvements in oligonucleotide characteristics. These include an increase in hybridization affinity toward a complementary RNA (1.5 degrees C per modification) and an increase in resistance toward both 3'-exonuclease and intracellular nucleases. These improvements result in a substantial increase in oligonucleotide potency (>20-fold after 72 h). The most active compound identified was used to examine the role played by protein kinase C-alpha in mediating the phorbol ester-induced changes in c-fos, c-jun, and junB expression in A549 lung epithelial cells. Depletion of protein kinase C-alpha protein expression by this oligonucleotide lead to a reduction in c-jun expression but not c-fos or junB. These results demonstrate that 2'-O-(2-methoxy)ethyl-modified antisense oligonucleotides are 1) effective inhibitors of protein kinase C-alpha expression, and 2) represent a class of antisense oligonucleotide which are much more effective inhibitors of gene expression than the widely used phosphorothioate antisense oligodeoxynucleotides.     

Parathyroid hormone-related protein expression in vascular smooth muscle of spontaneously hypertensive rats: evidence for lack of response to angiotensin II

OBJECTIVE: We studied the expression of parathyroid hormone (PTH)-related protein in vascular smooth muscle cells of spontaneously hypertensive rats (SHR) using Wistar-Kyoto (WKY) and Sprague-Dawley rats as normotensive controls. METHODS: Aortae from 4- and 18-week-old SHR versus age-matched WKY and Sprague-Dawley rats were excised to obtain total RNA or smooth muscle cells. The cells were subcultured in Dulbecco's Modified Eagle's Medium containing 10% fetal calf serum, then serum-deprived for 72 h and stimulated with 0.1 micromol/I angiotensin II. PTH-related protein, c-myc and angiotensin II type qa receptor (AT1aR) messenger (m)RNA levels were measured by Northern blot, using total RNA extracted by phenol/chloroform. The effects of PTH-related protein(1-34)NH2 intravenous injections on arterial blood pressure and the heart rate were studied in anesthetized SHR and WKY rats. RESULTS: The Northern blots showed a significantly higher abundance of PTH-related protein mRNA in aortae of SHR versus WKY rats in the prehypertensive state but no significant difference in adult animals. In cultured aortic smooth muscle cells, angiotensin II induced a four- to sixfold increase in PTH-related protein mRNA levels in smooth muscle cells from normotensive animals, but failed to elicit a significant response in smooth muscle cells derived from SHR in either the prehypertensive or the hypertensive state. This lack of response to angiotensin II in SHR smooth muscle cells was not due to decreased expression or responsiveness of the AT1aR, since SHR smooth muscle cells had more AT1aR mRNA than Sprague-Dawley smooth muscle cells, and angiotensin II-induced activation of c-myc was faster and greater in smooth muscle cells derived from 4- or 18-week-old SHR than in Sprague-Dawley smooth muscle cells. In contrast, PTH-related protein(1-34)NH2 induced a long-lasting dose-dependent hypotensive and tachycardic response in both SHR and WKY rats, indicating that SHR retained responsiveness to the vasodilator. CONCLUSIONS: PTH-related protein gene expression in response to angiotensin II is impaired in SHR arteries. A deficiency in this potent local vasodilator may contribute to the development and/or maintenance of arterial hypertension in this model.