We have studied the responses to electrical and chemical stimulation of the ventrolateral medulla in the chloralose-anesthetized, paralyzed, artificially ventilated rat. Locations of most active pressor responses were compared to regions containing neurons labeled immunocytochemically for phenylethanolamine N-methyltransferase (PNMT), the enzyme catalyzing the synthesis of adrenaline. Elevations of arterial pressure (+81.6 +/- 2.5 mm Hg) and cardioacceleration (+73 +/- 13.6 bpm) were elicited with low current (5 times threshold of 9.5 +/- 1.1 microA) electrical stimulation in a region of rostral ventrolateral medullary reticular formation we have termed the nucleus reticularis rostroventrolateralis (RVL). Electrical stimulation of the RVL increased plasma catecholamines (16.8-fold for adrenaline, 5.3-fold for noradrenaline, and 1.9-fold for dopamine) and vasopressin (1.7-fold before spinal transection, 4.7-fold after). The location of the most active pressor region in the ventrolateral medulla corresponded closely with the location of C1 adrenaline-synthesizing (PNMT-containing) neurons. In addition, the location of the most active pressor region in the dorsomedial medulla corresponded with the location of a bundle of PNMT-containing axons. Unilateral injections into the RVL of the excitatory amino acid monosodium L-glutamate (50 pmol to 10 nmol), but not saline, caused transient dose-dependent and topographically specific elevations (maximum +71.6 +/- 4.9 mm Hg) of arterial blood pressure and tachycardia. Injections of the rigid structural analogue of glutamate, kainic acid, caused large, prolonged (at least 15 min) pressor responses and tachycardia. Unilateral injections of the inhibitory amino acid gamma-aminobutyric acid (GABA) into the RVL caused transient dose-dependent hypotension (maximum -40.8 +/- 6.6 mm Hg) and bradycardia, whereas the specific GABA antagonist bicuculline caused prolonged (10 to 20 min) elevations (+64.2 +/- 6.8 mm Hg) of arterial pressure and tachycardia. By contrast, injections of the glycine antagonist strychnine had no significant effect. Bilateral injections of the neurotoxin, tetrodotoxin, dropped arterial pressure to low levels (51.7 +/- 4.7) not changed by subsequent spinal cord transection at the first cervical segment (52.5 +/- 6.2). We propose the following. (1) Neurons within the RVL, most probably C1 adrenaline-synthesizing neurons, exert an excitatory influence on sympathetic vasomotor fibers, the adrenal medulla, and the posterior pituitary. (2) These neurons are tonically active and under tonic inhibitory control, in part via GABAergic mechanisms--perhaps via the nucleus of the solitary tract (NTS).(ABSTRACT TRUNCATED AT 400 WORDS)

Anterograde, retrograde, and combined axonal transport methods were used to describe the descending efferent projections of a region of rostral ventrolateral medullary reticular formation important in cardiovascular control. We have termed this region, which contains C1 adrenaline-synthesizing neurons, the nucleus reticularis rostroventrolateralis (RVL). Efferent projections from the RVL innervate all segmental levels of the thoracic intermediolateral and intermediomedial columns as shown using retrograde transport of lectin-conjugated horseradish peroxidase (HRP) or fast blue dye, and anterograde transport of either HRP or labeled amino acids. The projection is highly specific in that there are no projections to thoracic dorsal or ventral horns. This innervation corresponds to the distribution of preganglionic sympathetic neurons in the intermediolateral column. In particular, terminals surround neurons projecting to the adrenal medulla, as demonstrated by combined anterograde and retrograde transport methods at the light level. Terminals containing phenylethanolamine-N-methyl transferase (PNMT) were mapped using immunocytochemical techniques. PNMT-labeled terminals were present at all levels of thoracic intermediolateral column, in a distribution similar to that of the descending projections from the RVL. We have previously shown using double label techniques (Ross et al., '81-'83), that many of the spinal projections of the RVL originate from C1 neurons. These data support our suggestion that certain bulbospinal neurons within the RVL, in particular the C1 neurons, are crucial for tonic vasomotor control.

Tyrosine hydroxylase [tyrosine monooxygenase, L-tyrosinetetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] was highly purified from rat caudate nuclei. When the pure hydroxylase was phosphorylated by incubation with cyclic AMP-dependent protein inase and[3]PJATP, 32P and tyrosine hydroxylase activity were detected after polyacrylamide gel electrophoresis in a single protein band. After sodium dodecyl sulfate gel electrophoresis,32P was detected only in a probable active subunit of tyrosine hydroxylase of molecular weight 62,000. Phosphorylation of the hydroxylase increased its activity by 2-fold, and was associated with an increase in Vm without any change in Km for either substrate or cofactor. We propose that the pool of native tyrosine hydroxylase is composed of a mixture of enzyme molecules in both active and probably inactive forms, that the active form is phosphorylated, and that phosphorylation produces an active form of the enzyme at the expense of an inactive one. An important, if not major, action of the cyclic nucleotides is to initiate the phosphorylation of intracellular proteins via the activity of protein kinases (1-3). This phosphorylation, which in many instances converts a biologically inert molecule into an active one, has been proposed to serve as a final common pathway for a variety of agents regulating cellular function (1-4). It remains to be established if phosphorylation is a mode of regulation of the activity of a class of proteins essential for neuronal function; namely, the enzymes that catalyze the biosynthesis of neurotransmitters.In brain, the enzyme tyrosine hydroxylase [tyrosine monooxygenase, L-tyrosine,tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating, EC 1.14.16.2] catalyzes the first and presumed rate-limiting step in the biosynthesis of the catecholamine neurotransmitters, dopamine and norepinephrine (5). Over the past several years the demonstration that the activity of the enzyme is altered by cyclic nucleotides (6-11) has raised the question as to whether Tyr hydroxylase may be regulated by phosphorylation, and if so, whether it is the enzyme itself which is phosphorylated. Subjection of impure enzyme, either partially purified or in tissue extracts, to conditions of phosphorylation has resulted in activation of the enzyme, attributed variously to an increased affinity of enzyme to cofactor (6, 9, 10) or reduction of inhibition by a natural inhibitor(s) (6, 12). However, since pure Tyr hydroxylase was not available, it was not possible to determine whether the molecule itself was directly phosphorylated (13) or whether the effect was due to phosphorylation of some other regulatory mechanism.In the present study, using highly purified Tyr hydroxylase, we have sought to determine if the enzyme can be directly phosphorylated. We shall demonstrate that the enzyme can be phosphorylated by a cyclic AMP (cAMP)-dependent protein kinase, that such phosphorylation increases the catalytic activity of the enzyme, and that the kinetics of acti...

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