Antagonists of 5-HT4 receptor-mediated responses in adult hippocampal neurons.

The study of serotonin-4 (5-HT4) receptors in the central nervous system has been hindered by the lack of effective, selective antagonists. However, recently, several novel compounds have been synthesized and shown to act as antagonists at 5-HT4 receptors in smooth muscle and embryonic neurons in culture. In the present study, intracellular electrophysiological recordings were used to test the effects of three of these compounds: endo-8-methyl-8-azabicyclo[3.2.1]oct-3-yl-2,3-dihydro-6-methoxy- 2-oxo-1H-benzimidazole-1-carboxylate (DAU 6285), [1-[2-(methylsulfonylamino)ethyl]-4-piperidinyl]methyl 1-methyl-1H-indole-3-carboxylate (GR 113808) and 2-diethylaminoethyl-(2-methoxy-4-amino-5-chloro) benzoate (SDZ 205-557) on the 5-HT4 reduction of the afterhyperpolarization seen in adult CA1 hippocampal neurons in brain slices. GR 113808, SDZ 205-557 and DAU 6285 all functioned as competitive antagonists at these 5-HT4 receptors. Although all three compounds tested acted as effective antagonists, they differed considerably in potency. When the potency of these antagonists at the 5-HT4 receptor that mediates the reduction of the afterhyperpolarization was compared with that observed for 5-HT4 receptors in biochemical and binding assays, an excellent correlation was observed. Among the antagonists tested, GR 113808 was the most potent (pA2 = GR 113808 > SDZ 205-507 > DAU 6285). It exhibited an apparent affinity for the 5-HT4 receptors in the low nanomolar range but did not antagonize 5-HT1A, beta-adrenergic or muscarinic receptor-mediated responses when applied at concentrations two orders of magnitude higher.(ABSTRACT TRUNCATED AT 250 WORDS)

Medial medullary contribution to tonic descending inhibition of visceral input.

In the present study, we sought to define the extent and source of tonic descending modulation of spinal neurons receiving visceral input from the afferent renal nerve (ARN). Spinal gray neurons responding to stimulation of the ARN in 64 chloralose-anesthetized rats were located primarily in laminae IV and V (70%), with fewer neurons located in laminae I and VII. ARN stimulation excited 76 and inhibited 8 neurons. Analysis of response latencies demonstrated that responses were due to activation of A delta- and/or C-fiber afferents. Reversible spinalization with cervical cold block (2-5 degrees C) affected activity in most neurons excited by ARN stimulation without affecting inhibited neurons. Cervical cold block increased the spontaneous activity of (disinhibited) the majority of neurons (54 of 76 neurons) and disfacilitated the spontaneous activity of 14 neurons. The evoked response to ARN stimulation was disinhibited by cold block in 51% and disfacilitated in 17% of the neurons, and there was a good correlation between neurons with disinhibited spontaneous activity and those with disinhibited evoked activity. Microinjections of muscimol (0.5-1 nmol) into the rostral medial medulla affected spontaneous and ARN-evoked activities similarly to cold block in 14 of 15 neurons, although the responses to muscimol were usually smaller in magnitude. We conclude that ARN input is modulated supraspinally and that the nucleus raphe magnus and adjacent neuropil contain neurons that contribute to tonic supraspinal inhibition of renal input in the rat.

Effects of electrical and chemical stimulation of nucleus raphe magnus on responses to renal nerve stimulation.

Electrical stimulation of the nucleus raphe magnus (NRM) inhibits some somatic and visceral input at the spinal level. This study was designed to examine the effects of electrical and chemical stimulation of NRM on neuronal responses to afferent renal nerve (ARN) stimulation. In chloralose-anesthetized rats, electrical stimulation of ARN elicited predominantly excitatory responses in spinal gray neurons. In 10 neurons studied, electrical stimulation of the NRM elicited an inhibition of spontaneous activity of 8 neurons and inhibited evoked responses to ARN stimulation in 6 neurons. Microinjection of glutamate (5-10 nmol in 0.5-1 microliter) into the NRM elicited an inhibition of spontaneous activity in 9 neurons, a facilitation in 6 neurons and no response in 8 neurons receiving ARN input. Responses evoked by ARN stimulation were inhibited in 12 neurons, facilitated in 4 neurons and not affected in 8 neurons. We conclude that renal input can be modulated at the spinal level by activation of the NRM and adjacent tissue. Furthermore, the inhibition of spinal gray neuronal responses elicited by stimulation of the NRM appears to be due, at least in part, to activation of fibers of passage since non-selective electrical stimulation is more efficacious than selective chemical stimulation of neuronal soma and dendrites.

Kappa-flavitoxin: isolation of a new neuronal nicotinic receptor antagonist that is structurally related to kappa-bungarotoxin.

A peptide, termed kappa-flavitoxin (kappa-flavitoxin), has been purified from the venom of the red-headed krait, Bungarus flaviceps, by low- and high-pressure liquid chromatography. kappa-Flavitoxin has a pI of 8.8 and an apparent molecular weight on sodium dodecyl-sulfate-polyacrylamide gel electrophoresis of 6500 Da. kappa-Flavitoxin is a potent inhibitor of nicotinic transmission in autonomic ganglia, producing a complete and long-lasting blockade at doses as low as 50 nM. Intracellular recordings reveal a selective blockade of neuronal nicotinic receptors by the toxin, with no effects on other active or passive properties of neuronal membranes. kappa-Flavitoxin shares a number of pharmacological and biochemical properties with kappa-bungarotoxin, purified from the venom of Bungarus multicinctus. The two peptides exhibit considerable homology in their amino acid sequences. Radiolabeled kappa-flavitoxin binds to a nicotinic site in ciliary ganglia previously identified by kappa-bungarotoxin, which appears to be associated with the neuronal nicotinic receptor. This site is not recognized by alpha-bungarotoxin, which also does not block nicotinic transmission in this ganglion.

Cyclic nucleotide phosphodiesterase isozymes in neuroblastoma cells.

Adenosine 3',5'-cyclic monophosphate (cAMP) content of neurons is determined not only by the rate of synthesis but also by the rate of hydrolysis by cyclic nucleotide phosphodiesterases. Multiple forms of cyclic nucleotide phosphodiesterase exist in brain and other tissues, and these may be regulated by various hormones and neuromodulators. The present study examines this regulation in a cloned line of neuroblastoma cells (N18TG2). A biphasic Lineweaver-Burk plot of cAMP hydrolysis revealed two Kms approximating 5 and 25 microM. Lineweaver-Burk plots of cGMP hydrolysis were linear over a range of 1 microM to 1 mM and exhibited a Km of 37 microM. Neither cAMP nor cGMP competed for hydrolysis of the alternative cyclic nucleotide. No evidence for an allosteric activation of cAMP phosphodiesterase by cGMP was found. Calcium regulation of phosphodiesterase was not found in spite of preparation of the cell extract with several protease inhibitors, and addition of exogenous calmodulin. No effect of calmodulin antagonists (calmidazolium, W7, or trifluoperazine) was observed in vitro or in situ. Growth of the cells in the presence of 200 nM 3,5,3'-triiodothyronine (T3) resulted in an increased hydrolysis of cAMP but of cGMP. This increase was attributed to an increase in Vmax with no change in either high or low Km. This response was blocked by cycloheximide, suggesting that the thyroid hormone effect requires protein synthesis. The thyroid hormone response in neuroblastoma cells is compared with the results of other studies of thyroid hormone effects on phosphodiesterase in other tissues in vivo.