Hexamethonium

Abstract: The experiments to be described have been concerned with the effect of vagus stimulation in the isolated heart and auricles of the rabbit. In the vagus-heart experiments the action of cocaine, of hexamethonium and of the cardiac glycoside ouabain was studied. As recently as 1953, Obrink & Essex state that 'it has become a widely accepted fact that stimulation of the vagi does not influence an isolated heart after 20-30 min. The reason for the transient response is not known.' Not many attempts to study this question have been found in the literature. Effects of vagal stimulation on perfused isolated heart preparations have been described by a number of workers (Cullis & Tribe, 1913; Middleton, 1947; Perry and Talesnik, 1953; Perry & Reinert, 1954), but of these only Middleton made a statement concerning the length of time his preparations worked satisfactorily. He perfused the cat's heart and found that vagal effects were obtained for 6 hr provided the heart remained in situ, but when the heart was isolated, vagal effects were not obtained after 10-30 min. Using the method now to be described it was possible to make observations for periods up to 9 hr.

Abstract: This study establishes that presynaptic nicotinic receptors modulate dopamine release in the mouse striatum. Nicotinic agonists elicit a dose-dependent increase in the release of [3H]dopamine from synaptosomes prepared from mouse striatum. At low concentrations, this release is Ca2+ dependent, whereas at higher concentrations Ca(2+)-independent, mecamylamine-insensitive release was also observed. The Ca(2+)-dependent nicotine-evoked release was not blocked by alpha-bungarotoxin but was effectively blocked by neuronal bungarotoxin as well as several other nicotinic receptor antagonists. The relationship between potency for stimulation of release for agonists and potency for inhibition of release for antagonists was compared to the affinity of these compounds for the [3H]nicotine binding site. The overall correlation between release and binding potency was not high, but the drugs may be classified into separate groups, each of which has a high correlation with binding. This finding suggests either that more than one nicotinic receptor regulates dopamine release or that not all agonists interact with the same receptor in an identical fashion.

Abstract: 1. A new smooth muscle preparation, the rat anococcygeus muscle, is described. The muscle is paired, thin, consists of smooth muscle only and the muscle cells are organized in parallel bundles. It has a dense adrenergic innervation distributed throughout the muscle but apparently no cholinergic innervation. The muscles are easily isolated.2. The muscle contracts to noradrenaline, acetylcholine, furmethide, 5-hydroxytryptamine, but not to histamine. Isoprenaline produces contraction at high concentrations. The effects of noradrenaline and acetylcholine are blocked by phentolamine and atropine respectively. The response to isoprenaline is little affected by propranolol.3. The muscle contracts in response to field stimulation or stimulation of extrinsic nerves. This response is completely blocked by phentolamine but unaffected by hexamethonium or atropine.4. Guanethidine 10(-6)-5 x 10(-6)M blocks the motor response to nerve stimulation and potentiates that to noradrenaline. Higher concentrations of guanethidine raise tone. In the presence of raised tone, field stimulation produces an inhibitory response insensitive to hexamethonium but abolished by tetrodotoxin 2 x 10(-7) g/ml. This inhibitory response to stimulation can also be shown after other drugs which raise tone.5. The inhibitory response to nerve stimulation is not mimicked by acetylcholine, isoprenaline or ATP, nor blocked by atropine, phentolamine, phenoxybenzamine, propranolol, hexamethonium or lysergic acid diethylamide.

Abstract: Using functional co-cultures of rat carotid body (CB) O2 chemoreceptors and ‘juxtaposed’ petrosal neurones (JPNs), we tested whether ATP and ACh acted as co-transmitters. Perforated-patch recordings from JPNs often revealed spontaneous and hypoxia-evoked (PO2≈5 mmHg) excitatory postsynaptic responses. The P2X purinoceptor blocker, suramin (50 μM) or a nicotinic ACh receptor (nAChR) blocker (hexamethonium, 100 μM; mecamylamine, 1 μM) only partially inhibited these responses, but together, blocked almost all activity. Under voltage clamp (-60 mV), fast perfusion of 100 μM ATP over hypoxia-responsive JPNs induced suramin-sensitive (IC50 = 73 μM), slowly-desensitizing, inward currents (IATP) with time constant of activation τon = 30.6 ± 4.8 ms (n = 7). IATP reversed at 0.33 ± 3.7 mV (n = 4), and the dose-response curve was fitted by the Hill equation (EC50 = 2.7 μM; Hill coefficient ≈0.9). These purinoceptors contained immunoreactive P2X2 subunits, but their activation by α,β-methylene ATP (α,β-meATP; EC50 = 2.1 μM) suggests they are P2X2/P2X3 heteromultimers. Suramin and nAChR blockers inhibited the extracellular chemosensory discharge in the intact rat carotid body-sinus nerve preparation in vitro. Further, P2X2 immunoreactivity was widespread in rat petrosal ganglia in situ, and co-localized in neurones expressing the CB chemo-afferent marker, tyrosine hydroxylase (TH). P2X2 labelling in the CB co-localized with nerve-terminal markers, and was intimately associated with TH-positive type 1 cells. Thus ATP and ACh are co-transmitters during chemotransduction in the rat carotid body. In mammals, an early step in the initiation of compensatory reflex hyperventilation during blood hypoxaemia appears to involve neurotransmitter release from O2-sensitive chemoreceptor (type 1) cells in the carotid body onto apposed sensory nerve endings (reviewed by Gonzalez et al. 1994). Excitation of these endings by released transmitter(s) is proposed to result in the increased afferent spike discharge in the carotid sinus nerve, whose chemo-afferent cell bodies are located in the petrosal ganglion. A long-standing, unresolved issue is the identity of the neurotransmitter(s) initiating this reflex (Gonzalez et al. 1994; Fitzgerald et al. 1997). Whereas the best studied carotid body neurotransmitter dopamine has recently fallen into disfavour (e.g. Donnelly, 1996), there has been a rekindling of interest in ACh as a major carotid body neurotransmitter during chemo-excitation, even though it too has had a long and controversial history (Gonzalez et al. 1994; Fitzgerald et al. 1997; Nurse & Zhang, 1999). In order to probe these transmitter mechanisms, we recently developed an attractive co-culture preparation consisting of dispersed rat type 1 cell clusters and dissociated petrosal neurones (Zhong et al. 1997; Nurse & Zhang, 1999). The main advantages are that: (i) functional connections develop de novo; (ii) subthreshold synaptic events can be conveniently studied by recording from petrosal somas, juxtaposed to type 1 cell clusters; and (iii) drugs have relatively unrestricted access to the synaptic sites under these monolayer conditions. Using this preparation we recently provided strong evidence for ACh as a co-released transmitter from type 1 cells during chemosensory signalling (Zhong et al. 1997; Nurse & Zhang, 1999). We further characterized the properties of the nicotinic ACh receptors (nAChR) expressed by petrosal neurones using pharmacological and electrophysiological techniques (Zhong & Nurse, 1997). However, it was evident that ACh alone could not account entirely for the hypoxia-induced ‘postsynaptic’ responses recorded in co-cultured neurones. We therefore considered the possibility that other co-released transmitters might be involved. An attractive candidate for an excitatory, sensory co-transmitter in carotid body function is ATP. First, the excitatory effects of ATP on carotid body chemosensory fibres have been recognized for some time, based on sinus nerve recordings during intracarotid injections of ATP (Jarish et al. 1952; Spergel & Lahiri, 1993). Second, ATP is frequently co-released with ACh in several neuronal preparations (Schweitzer, 1987; Bean, 1992; Zimmerman, 1994; Silinsky & Redman, 1996), and purinoceptors are widely distributed on sensory neurones and their terminals (Khakh et al. 1995; Lewis et al. 1995; Vulchanova et al. 1996; Wildman et al. 1997). Third, cation-selective P2X purinoceptor channels were first described in the peripheral nervous system, where they appear to be important for mediating fast excitatory neurotransmission at a variety of synapses (Surprenant et al. 1995). In the present study we identified an important role for ATP as a co-transmitter in carotid body function, based largely on the sensitivity of the afferent postsynaptic responses to the P2 purinoceptor blocker suramin. However, since suramin blocks both ligand-gated P2X and G-protein-coupled P2Y purinoceptors (Dunn & Blakely, 1988; Evans et al. 1992), we carried out a more detailed characterization of the P2 receptor subtype(s) expressed by chemosensory petrosal neurones using electrophysiological, pharmacological and immunofluorescence techniques. In addition, in order to validate the results obtained from co-cultures, we compared the effects of both nicotinic cholinergic and purinoceptor blockers on the afferent discharge recorded from the intact rat carotid body sinus nerve preparation in vitro (Pepper et al. 1995; Donnelly, 1996). These combined data led to the conclusion that co-release of ATP and ACh, acting at postsynaptic P2X2-containing and nicotinic-ACh receptors, respectively, plays a dominant role in rat carotid body PO2 chemotransmission.