Suzanne Killinger

LONG-TERM REGULATION OF ARTERIAL BLOOD PRESSURE BY HYPOTHALAMIC NUCLEI: SOME CRITICAL QUESTIONS

1. The long‐term level of arterial pressure is dependent on the relationship between arterial pressure and the urinary output of salt and water, which, in turn, is affected by a number of factors, including renal sympathetic nerve activity (RSNA). In the present brief review, we consider the mechanisms within the brain that can influence RSNA, focusing particularly on hypothalamic mechanisms.

Cardiovascular Responses Evoked by Leptin Acting on Neurons in the Ventromedial and Dorsomedial Hypothalamus

Abstract—Leptin, a circulating hormone produced by adipose tissue, is believed to act on the hypothalamus to increase sympathetic vasomotor activity, in addition to its well-known effects on appetite and energy expenditure. In this study, we determined the cardiovascular effects of direct application of leptin to specific cell groups within the hypothalamus that are known to be activated by circulating leptin. In rats anesthetized with urethane, microinjections of leptin (16 ng in 20 nL solution) were made into the ventromedial hypothalamic nucleus, dorsomedial hypothalamic nucleus, and paraventricular nucleus. Compared with vehicle solution, microinjections of leptin into the ventromedial hypothalamic nucleus evoked significant increases in arterial pressure and renal sympathetic nerve activity, but not heart rate. In contrast, microinjections of leptin into the dorsomedial hypothalamic nucleus evoked significant increases in arterial pressure and heart rate but not renal sympathetic nerve activity, whereas microinjections of leptin into the paraventricular nucleus had no significant effect on any of the measured cardiovascular variables. These results indicate that the ventromedial and dorsomedial hypothalamic regions might be important sites at which leptin activation leads to increases in sympathetic vasomotor activity and heart rate, as occurs in obesity-related hypertension.

Autoradiography of P2X ATP receptors in the rat brain

1 Binding of a P2, receptor specific radioligand, [3H]‐α,β‐methylene adenosine triphosphate ([3H]‐m43‐MeATP) to sections of rat brain was reversible and association/dissociation parameters indicated that it consisted of two saturable components. Non‐specific binding was very low (<7% at 10 nM ligand concentration). 2 The binding was completely inhibited by suramin (IC50∼ 14–26 μm) but none of the ligands specific for P2y receptors such as 2‐methylthio‐adenosine triphosphate (2‐methyl‐S‐ATP) and 2‐chloro‐adenosine triphosphate (2‐C1‐ATP) nor 2‐methylthio‐adenosine diphosphate (2‐methyl‐S‐ADP) a ligand for the P2 receptor on blood platelets (′P2T′ type) produced strong inhibitions except for P1, P4‐di(adenosine‐5′)tetraphosphate (Ap4A). 3 Inhibitors of Na+, K+‐dependent adenosine triphosphatase (ATPase) ouabain, P1‐ligand adenosine and an inhibitor of transport of, respectively, adenosine and cyclic nucleotides, dilazep, had no effect. 4 The highest density of P2x binding sites was found to be in the cerebellar cortex but the binding sites were present in all major brain regions, especially in areas known to receive strong excitatory innervation.

Effects of l-trans-pyrrolidine-2,4-dicar☐ylate and l-threo-3-hydroxyaspartate on the binding of [3H] l-aspartate, [3H]α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), [3H] dl-(E)-2-amino-4-propyl-5-phosphono-3pentenoate (cgp 39653), [3H]6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) [3H]kainate studied by autoradiography in rat forebrain

l-trans-Pyrrolidine-2,4-dicar☐ylate (l-t-PDC) and L-threo-3-hydroxyaspartate (l-t-30HA), compounds known to interact strongly with the Na+-dependent high affinity uptake of excitatory amino acids in central nervous tissue, were tested as potential inhibitors of binding to glutamate receptors and transport sites in frozen sections of rat brain. [3H]α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), [3H]6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and [3H]kainate were used as ligands for the binding sites on the “non-NMDA” classes of glutamate receptors and [3H] dl-(E)-2-amino-4-propyl-5-phosphono-3-pentenoate (CGP 39653) was used to label NMDA receptor binding sites. The Na+-dependent glutamate-uptake site was marked by [3H] l-aspartate. The autoradiograms, obtained by exposing3H-sensitive film to sections of rat forebrain preincubated with3H-labelled ligands, were scanned by laser beam and quantified. Distribution patterns of the receptor and transporter sites visualized by the3H-labelled ligands were compatible with previously published results. [3H]CNQX binding, however, was found to be significantly decreased by Na+l-t-30HA was about an order of magnitude stronger than l-t-PDC as an inhibitor of [3H] l-aspartate binding. Neither of the compounds had any important effect at the “non-NMDA” receptor binding sites but l-t-30HA was a weak inhibitor of [3H]CGP 39653 binding (<40% at 100 μ M). The results suggest that, at low nanomolar concentrations, both compounds are likely to be selective for Na+-dependent high affinity glutamate transporter sites. Moreover, l-t-30HA seems to have a sufficiently high affinity for the site to be almost certainly useful, if available in a3H-labelled form, as a ligand in autoradiographic studies.

Angiotensin II evokes hypotension and renal sympathoinhibition from a highly restricted region in the nucleus tractus solitarii

Microinjections of low doses (in the femtomolar or low picomolar range) of angiotensin II (Ang II) into the nucleus tractus solitarii (NTS) evoke depressor responses. In this study we have mapped in the rat the precise location of the subregion within the NTS at which Ang II evokes significant sympathoinhibitory and depressor responses. Microinjections of 1 pmol of Ang II evoked large decreases (>or=20% of baseline) in renal sympathetic nerve activity (RSNA), from a highly restricted region in the medial NTS, at or very close to the level 0.2 mm caudal to the obex. Microinjections of the same dose of Ang II into the commissural or lateral NTS at the same rostrocaudal level, or into the medial and lateral NTS at the level of the obex evoked significantly smaller sympathoinhibitory responses, while microinjections into more rostral or caudal levels of the NTS evoked significant sympathoinhibitory responses even less frequently. In most cases (71%), the sympathoinhibitory responses were accompanied by depressor responses, the magnitudes of which were also greater within the medial NTS at the level 0.2 mm caudal to obex, as compared to the surrounding subregions. The results demonstrate that the cardiovascular effects of Ang II in the NTS are highly site-specific. Taken together with previous studies, the results also indicate that the neurons in the NTS that mediate the Ang II-evoked sympathoinhibition are a discrete subgroup of the population of sympathoinhibitory neurons within the nucleus.

Renal sympathetic and cardiac changes associated with anaphylactic hypotension.

Severe anaphylactic reactions can result in life-threatening hypotension, but little is known about the autonomic changes that accompany the hypotensive response. The aim of this study was to determine the renal sympathetic and cardiac responses to anaphylactic hypotension, and to evaluate the contribution of sinoaortic and vagal afferent inputs in producing these responses. Rats were sensitized with bovine serum albumin (BSA) and, after 10-14 days, were anaesthesized with sodium pentobarbitone and arterial pressure, heart rate (HR), and renal sympathetic nerve activity (RSNA) were recorded. In about two thirds of the rats, injection of BSA evoked a severe and sustained hypotension, while in the remainder, there was either a more transient hypotension or else no significant change in arterial pressure. In control unsensitized rats, BSA injection had no significant effect on arterial pressure, heart rate, or RSNA. The BSA-induced hypotension in sensitized rats was associated with increases in HR and RSNA, the magnitudes of which were correlated with the magnitude of the hypotension. There were two components to the cardiac and renal sympathoexcitatory response: (1) an initial increase in HR and RSNA, which immediately followed the onset of hypotension and which was abolished by sinoaortic denervation and vagotomy, and (2) a delayed and gradual increase in HR and RSNA, which continued even while the arterial pressure was recovering and was reduced but not abolished by sinoaortic denervation and vagotomy. Thus, BSA-induced anaphylactic hypotension causes prolonged tachycardia and renal sympathoexcitation, which is only partly due to reflex effects arising from sinoaortic baroreceptors and cardiopulmonary receptors.

Autoradiographic studies indicate regional variations in the characteristics of l-glutamate transporters in the rat brain

Quantitative autoradiography of [3H]L-aspartate binding in thaw-mounted sections of rat brain has shown that L-trans-pyrrolidine-2,4-dicarboxylate and D-threo-3-hydroxyaspartate but not DL-2 aminoadipate strongly interacted with the binding sites while dihydrokainate, kainate and beta-aminoadipate produced only weak effects. The potency of inhibitors did not vary from one region to another in the telencephalon (neocortex, hippocampus and neostriatum) but, D-threo-3-hydroxyaspartate, L-trans-pyrrolidine-2,4-dicarboxylate, kainate and dihydrokainate inhibited [3H]L-aspartate binding in the cerebellar cortex less potently than that in the forebrain. Characteristics of the known excitatory amino acid transporters can, in part, explain the present results but contributions from additional transporter molecules to the heterogeneity of [3H]L-aspartate binding sites cannot be ruled out.

Sensitivity of the binding sites on glutamate transporters to neurotoxic agents

Autoradiography of [3H]L-aspartate binding to sections of rat brain was used to study the sensitivity of Na(+)-dependent glutamate transporters to neurotoxic agents such as Zn2+, NH4+, oxygen-containing free radicals and mercuric chloride. Only mercuric chloride was a strong inhibitor in cerebral neocortex, hippocampus, neostriatum, thalamus and cerebellar cortex. It is concluded that the substrate-binding sites on Na(+)-dependent glutamate transporters are relatively resistant to direct effects of Zn2+, NH4+ and free radicals but they may depend on the structural integrity of thiol bonds. Direct inhibitory effect of mercury on the binding site could significantly contribute to its long-term neurotoxicity.

Activation of 5-hydroxytryptamine-1A receptors suppresses cardiovascular responses evoked from the paraventricular nucleus

Activation of central 5-hydroxytryptamine-1A (5-HT(1A)) receptors powerfully inhibits stress-evoked cardiovascular responses mediated by the dorsomedial hypothalamus (DMH), as well as responses evoked by direct activation of neurons within the DMH. The hypothalamic paraventricular nucleus (PVN) also has a crucial role in cardiovascular regulation and is believed to regulate heart rate and renal sympathetic activity via pathways that are independent of the DMH. In this study, we determined whether cardiovascular responses evoked from the PVN are also modulated by activation of central 5-HT(1A) receptors. In anesthetized rats, the increases in heart rate and renal sympathetic nerve activity evoked by bicuculline injection into the PVN were greatly reduced (by 54% and 61%, respectively) by intravenous administration of (±)-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), an agonist of 5-HT(1A) receptors, but were then completely restored by subsequent administration of WAY-100635, a selective antagonist of 5-HT(1A) receptors. Microinjection of 8-OH-DPAT directly into the PVN did not significantly affect the responses to bicuculline injection into the PVN, nor did systemic administration of WAY-100635 alone. In control experiments, a large renal sympathoexcitatory response was evoked from both the PVN and DMH but not from the intermediate region in between; thus the evoked responses from the PVN were not due to activation of neurons in the DMH. The results indicate that activation of central 5-HT(1A) receptors located outside the PVN powerfully inhibits the tachycardia and renal sympathoexcitation evoked by stimulation of neurons in the PVN.