Antonio Granata

AN ENDOGENOUS CLONIDINE-DISPLACING SUBSTANCE FROM BOVINE BRAIN: RECEPTOR BINDING AND HYPOTENSIVE ACTIONS IN THE VENTROLATERAL MEDULLA

A substance has been isolated from bovine brain which displaces 3H-clonidine binding to rat brain membranes (clonidine-displacing substance; CDS). To determine whether CDS is similar to the antihypertensive agent clonidine, the in vitro binding properties of partially-purified CDS and its physiological action in the rostral ventrolateral medulla were examined. Like clonidine, CDS potently inhibited 3H-para-aminoclonidine binding to receptors in bovine ventrolateral medulla membranes (clonidine, IC50 = 24 +/- 8nM; CDS, IC50 = 0.30 +/- .10 Units), with highest affinity for non-adrenergic sites (clonidine, IC50 = 6 +/- 1nM; CDS, IC50 = 0.12 +/- .07 Units). CDS had no effect at beta-adrenergic or muscarinic cholinergic receptors. Like clonidine, CDS elicited a potent, reversible (less than 10 min) dose-dependent fall in arterial pressure (AP) and heart rate when microinjected specifically into the C1 area of the rostral ventrolateral medulla in the rat (maximum delta AP, -65 +/- 7 mm Hg). CDS represents an as-yet-uncharacterized endogenous, physiologically-active agent in brain which may participate in cardiovascular control via non-adrenergic receptors in the rostral ventrolateral medulla.

A1 noradrenergic neurons tonically inhibit sympathoexcitatory neurons of C1 area in rat brainstem

In rats anesthetized with urethane and paralyzed, bilateral microinjections of kainic acid (KA) into the region of caudal ventrolateral medulla (CVL) containing noradrenergic neurons of the A1 group (A1 area) elicited a decrease followed by an increase in arterial pressure (AP), heart rate (HR) and sympathetic renal nerve activity (RNA). The sympathoinhibitory and sympathoexcitatory effects of KA were prevented by bilateral microinjection of tetrodotoxin into an area of the rostral ventrolateral medulla (RVL) containing C1 adrenergic neurons (the C1 area). In contrast, the autonomic responses were not altered by interruption of the two other principal projections of A1 area neurons, namely to the hypothalamus or to the nucleus tractus solitarii. Bilateral microinjections of tyramine, clonidine, alpha-methylnoradrenaline or histamine into the C1 area elicited a dose-dependent, anatomically specific and reversible decrease in AP, HR and RNA. The effect of tyramine was blocked by previous microinjection of reserpine, 6-hydroxydopamine (6-OHDA), or phentolamine into the C1 area. Pretreatment with phentolamine unveiled a hypertensive effect of alpha-methylnoradrenaline. All effects of alpha-methylnoradrenaline were blocked by pretreatment of the C1 area with phentolamine plus DL-propranolol, whereas those elicited by histamine prevailed. Pretreatment of the C1 area with 6-OHDA abolished all changes in AP and HR elicited by microinjections of KA into the A1 area. We conclude that (1) neurons of the CVL tonically inhibit sympathetic activity, (2) this effect is mediated by an action upon vasomotor neurons of the C1 area of RVL, (3) the inhibition is mediated by noradrenergic projections from A1 neurons into the C1 area, and (4) this tonic sympathoinhibitory effect is independent of the baroreceptor reflex.

Evidence that glutamic acid is the neurotransmitter of baroreceptor afferents terminating in the nucleus tractus solitarius (NTS)

The possibility that L-glutamic acid (L-Glu) might be the neurotransmitter of baroreceptor afferents terminating in the nucleus tractus solitarius (NTS) and affecting activity of the autonomic nervous system was tested using a variety of techniques. Effects on blood pressure and heart rate of microinjections of L-Glu and antagonists into the NTS supported this contention. Use of blocking agents was confirmatory. Biochemical studies provided evidence that the agent (glutamic acid) is synthesized, stored and reaccumulated in these nerve endings. Furthermore, it was shown that L-Glu is released from the NTS in vivo. Degeneration of afferent terminals due to removal of the nodose ganglia reduced L-Glu levels in the NTS. After discussion of this and additional evidence that L-Glu is the transmitter and a consideration of reasons for eliminating other possibilities (substance P, etc.) it was concluded that L-glutamic acid is the neurotransmitter of baroreceptor afferents.

Role of Adrenaline Neurons of Ventrolateral Medulla (The C1 Group) in the Tonic and Phasic Control of Arterial Pressure

We have sought to determine if adrenaline neurons of the C1 group are responsible for cardiovascular functions heretofore attributed to neurons in the rostral ventrolateral medulla. C1 neurons were identified in rat with antibodies to the adrenaline synthesizing enzyme, PNMT. These project to spinal cord wherein they selectively innervate the sympathetic columns. C1 neurons are also innervated by projections, mostly unilateral, from the nucleus tractus solitarii (NTS). Stimulation of the C1 area electrically, by local injection of the excitatory amino acid, L-glutamate, or with the GABA antagonist bicuculline, elevates arterial pressure (AP). Bilateral electrolytic lesions, microinjection of GABA, or administration of tetrodotoxin, in contrast, collapses AP to levels comparable to that of spinal cord transection. After lesions of one NTS, a lesion of the contralateral C1 area abolishes all reflex activity elicited by electrical or natural stimulation of baroreceptors on the side of C1 lesion without modifying resting AP. Lesions of axon bundles of PNMT neurons in the medulla also abolish baroreflexes after unilateral NTS lesions. C1 neurons appear to be the neurons mediating cardiovascular effects of application of drugs or cold to rostral portions of the ventrolateral medulla. We conclude adrenaline neurons of the C1 area represent the purportedly tonic vasomotor neurons of the rostral ventrolateral medulla and mediate the vasodepressor limb of reflexes arising from arterial baroreceptors and other cardiopulmonary afferents. Whether the tonic vasomotor response to stimulation of C1 neurons is dependent upon the release of adrenaline is not yet certain.

Hypotensive action of clonidine analogues correlates with binding affinity at imidazole and not alpha-2-adrenergic receptors in the rostral ventrolateral medulla.

Clonidine acts within the rostral ventrolateral medulla to lower arterial pressure. The receptor mechanism for this action is unknown. In the rostral ventrolateral medulla clonidine binds not only to the ap2-adrenergic receptor but also to a novel class of sites which are specific for imidazolines and imidazoles and are distinct from adrenergic or histaminergic receptors. In order to distinguish whether a putative imidazole receptor or the aP2-receptor mediates the clonidine hypotensive response, a series of clonidine analogues were tested (1) in radioligand binding assays of their affinity at imidazole and aP2-adrenergic receptors, and (2) by microinjection into the rostral ventrolateral medulla of anaesthetized rats to measure their capacity to lower arterial pressure. The hypotensive response elicited by the test agents was strongly correlated with affinity at imidazole sites (r = 0.92) but not with aP2-adrenergic affinity (r = 0.36). An imidazole receptor in the rostral ventrolateral medulla may participate in the hypotensive action of clonidine.

Release of [3H]l-glutamine acid (l-Glu) and [3H]d-aspartic acid (d-Asp) in the area of nucleus tractus solitarius in vivo produced by stimulation of the vagus nerve

We have investigated the effects of bilateral electrical stimulation of the vagus nerves in anesthetized, paralyzed rats on the release of exogenously administered [3H]L-glutamic acid ([3H]L-Glu) or [3H]D-aspartic acid ([3H]D-Asp) from the intermediate portion of the nucleus tractus solitarius (NTS). Electrical stimulation of afferent fibers with the frequency, pulse, duration, and intensity required to activate C-fibers, elicited hypotension and bradycardia. Such stimuli induced the release of [3H]L-Glu, or its stable analogue [3H]D-Asp, from the NTS into perfusate collected through push-pull cannulae. The release of radioactive materials, calculated as a percent of increase in radioactivity above the prestimulation level, was for [3H]L-Glu 114.4 +/- 25.1% (n = 20) during bilateral vagal stimulation, and 45.6 +/- 11.3% (n = 9) (P less than 0.001) during unilateral stimulation. The release of [3H]D-Asp induced by bilateral vagal stimulation was 100.4 +/- 31.9%. The release, which was anatomically specific and restricted to the NTS, was directly related to stimulus (and hence reflex) intensity. Overflow of the inert substances [14C]urea OR [14C]sucrose, co-administered with the [3H] amino acids, did not increase at the same time. Local depolarization of the cells in the NTS by K+ (53mM) increased the overflow of [3H]L-Glu, as well as [14C]urea, and was able to induce the release of [3H]L-Glu when electrical stimulation failed to have an effect. The results are consistent with the hypothesis that L-Glu is a neurotransmitter of neurons in the NTS mediating vasodepressor response from vagal afferents, including those from systemic baroreceptors.

Sympathoinhibition by A1-noradrenergic neurons is mediated by neurons in the C1 area of the rostral medulla☆

In anesthetized, paralyzed rats, bilateral microinjections of kainic acid (KA) into an area of caudal ventrolateral medulla containing A1 noradrenergic neurons of the A1 group (A1 area) first reduced and then elevated arterial pressure (AP), heart rate (HR) and sympathetic renal nerve activity. These effects are attributable to initial excitation and then paralysis of local neurons by KA. The microinjection of tetrodotoxin into an area of rostral ventrolateral medulla containing adrenaline neurons of the C1 group (C1 area) and which is innervated by neurons in A1 area abolished all the effects of KA. The pretreatment of the C1 area with 6-hydroxy-dopamine (6-OHDA) also abolished all the effects of KA. In contrast interruption of projections of the A1 area to the hypothalamus or nucleus tractus solitarii had no effects. The latency for response evoked in renal nerve by stimulation of A1 area was about 10 ms longer than that elicited from C1 area. Tyramine microinjected into the C1 area elicited a dose-dependent decrease in AP prevented by local application of desmethylimipramine. We conclude that neurons of the A1 area tonically inhibit sympathetic activity by inhibiting neurons of the C1 area probably by release of NA.

Contributions of Arginine Vasopressin and the Sympathetic Nervous System to Fulminating Hypertension After Destruction of Neurons of Caudal Ventrolateral Medulla in the Rat

We sought to determine the role in control of arterial pressure (AP) of the caudal ventrolateral medulla (CVL), a region containing noradrenergic neurons of the A1 group. Electrical stimulation of the A1 area in anaesthetized rats elicits a fall of AP and heart rate at low, but not high, stimulus frequencies. Electrolytic lesions of the A1 area produce fulminating hypertension and a 13-fold increase in plasma arginine vasopressin (AVP). A1-hypertension is attenuated by treatment with an AVP antagonist and is also diminished in rats of the Brattleboro strain. After AVP blockade, the residual hypertension is abolished by treatment with ganglionic or alpha adrenergic blockers, but not by adrenalectomy. We conclude that the region of the CVL containing the A1 noradrenergic cells tonically inhibits the discharge of sympathetic nerve activity as well as the release of AVP from the pituitary.

In vivo release by vagal stimulation of L-[3H] glutamic acid in the nucleus tractus solitarius preloaded with L-[3H] glutamine

In anesthetized and paralyzed rats, using a push-pull perfusion technique, we examined the effect of bilateral vagal stimulation on the release of L-[3H] glutamic acid (L- [3H] Glu) from the nucleus tractus solitarius (NTS), after preloading the tissue either with L-[3H] Glu or L-[3H] glutamine (L- [3H] Gln). Vagal stimulation sufficient to produce a maximum fall of arterial pressure (AP) evoked release of L- [3H] Glu from the NTS when the tissue was preloaded with either 3H-Glu or 3H-Gln, and of D-[3H] aspartic acid (D-[3H] Asp) when this stable Glu analogue was used to preloaded with either 3H-Glu or 3H-Gln, and of D-[3H] precursor L-Gln is a good marker of the releasable pool of L-Glu in vivo and are consistent with the hypothesis that L-[3H] Glu is a neurotransmitter in the NTS, mediating the vasodepressor response from cardiopulmonary mechanoreceptors.

Hypotension and bradycardia elicited by histamine into the C1 area of the rostral ventrolateral medulla

In rat, anaesthetized with urethane and paralyzed, bilateral microinjections of histamine or naphazoline into a region of the rostral ventrolateral medulla (RVL) containing adrenergic neurons of the C1 group (C1 area) elicited a dose-dependent, anatomically specific and reversible decrease of arterial pressure (AP) and heart rate (HR). Similar effects were observed with the histamine agonists 4-imidazole acetic acid or the selective H2 agonist dimaprit. The selective H2 antagonist metiamide but not the H1 antagonist chlorpheniramine injected bilaterally into the C1 area reversibly blocked the hypotension and bradycardia elicited by histamine or dimaprit without affecting the responses produced by naphazoline. Neither metiamide nor chlorpheniramine change resting AP or HR. Pretreatment of the C1 area with the non-imidazole alpha-adrenoceptor blocker phenoxybenzamine while increasing resting AP and HR, antagonized the decrease of AP and HR elicited by the indirect sympathomimetic agonist tyramine but not those by histamine. We conclude, that histaminergic and tonic noradrenergic mechanisms coexist in the C1 area of the RVL mediating sympathoinhibitory responses, the effect of histamine is mediated by H2 but not by H1 receptors.