Mohammad A. Khoyi

Loss of Enteric Motor Neurotransmission in the Gastric Fundus of Sl/Sld Mice

Studies of W/WV mice, which lack intramuscular interstitial cells of Cajal (IC‐IM), have suggested that IC‐IM act as mediators of enteric motor neurotransmission in the gastrointestinal tract. We have studied Sl/Sld mice, which lack the ability to make membrane‐bound stem cell factor, to determine the consequences of inappropriate stem cell factor expression on IC‐IM populations and on enteric motor neurotransmission. IC‐IM were found within the circular and longitudinal muscles of the gastric fundus of wild‐type mice. IC‐IM were intimately associated with motor nerve terminals and nerve varicosities formed synaptic structures with these cells. IC‐IM were also connected with neighbouring smooth muscle cells via gap junctions. Immunohistochemistry and electron microscopy showed that IC‐IM were absent from fundus muscles of Sl/Sld mice, but the density of excitatory and inhibitory nerves was not significantly different than in wild‐type muscles. Loss of IC‐IM was associated with decreased membrane noise (unitary potentials) and significant reductions in post‐junctional excitatory and inhibitory enteric nerve responses. Reductions in neural responses were not due to defects in smooth muscle cells as responses to exogenous ACh and K+‐induced depolarization were normal in Sl/Sld mice. Responses to neurally released ACh were revealed in Sl/Sld mice by inhibiting ACh breakdown with the acetylcholinesterase inhibitor neostigmine. Inhibitory nerve stimulation elicited inhibitory junction potentials (IJPs) and relaxations in wild‐type mice. IJPs were reduced in amplitude and relaxation responses were absent in Sl/Sld mice. These observations suggest that membrane‐bound stem cell factor is essential for development of IC‐IM and that the close, synaptic‐like relationship between nerve terminals and IC‐IM may be the primary site of innervation by enteric motor neurons in gastric muscles.

Effect of the dihydropyridine Bay K 8644 on the release of [3H]-noradrenaline from the rat isolated vas deferens.

1 The effects of Bay K 8644 on the release of [3H]‐noradrenaline evoked by potassium, electrical stimulation or tyramine from the rat isolated vas deferens labelled with [3H]‐noradrenaline were investigated. 2 Bay K 8644 (1 μM) by itself did not affect the spontaneous release of tritium from the rat isolated vas deferens. However, it increased the calcium‐dependent release of tritium elicited by both high potassium (59 mM) and electrical field stimulation. 3 The exposure of rat vas deferens to phentolamine (10 μM) increased the release of tritium induced by potassium (59 mM) and electrical field stimulation. Bay K 8644 (1 μM) failed to increase further the release of tritium elicited by both stimuli in preparations previously treated with phentolamine (10 μM). 4 The calcium‐independent release of [3H]‐noradrenaline evoked by tyramine (10 μM) was not affected by Bay K 8644 (1 μM). 5 The results of our study support the view that α2‐adrenoceptors modulate noradrenaline release by restricting calcium influx into sympathetic nerve terminals through voltage‐dependent channels.

[Ca2+]i-sensitive, IP3-independent Ca2+ influx in smooth muscle of rat vas deferens revealed by procaine

1 The actions of procaine (10 mm) on noradrenaline‐induced effects on 45Ca‐influx, 45Ca‐efflux, 45Ca‐content, total inositol phosphates, inositol‐1,4,5‐trisphosphate, and contractile status of the rat vas deferens were examined. 2 Noradrenaline alone had no effect on 45Ca‐influx or 45Ca‐content, but released Ca2+ from intracellular stores as indicated by an increased 45Ca‐efflux and increased total inositol phosphates, specifically inositol‐1,4,5‐trisphosphate, leading to contraction of the rat vas deferens. 3 Noradrenaline, in the presence of 10 mm procaine, increased 45Ca‐influx and 45Ca‐content. Procaine blocked the noradrenaline‐induced 45Ca‐efflux, the increase in total inositol phosphates, the increase in inositol‐1,4,5‐trisphosphate, and contraction. 4 The noradrenaline‐induced increase in 45Ca influx which was observed in the presence of procaine was abolished by phentolamine and nifedipine but was not altered significantly by propranolol suggesting that, in the presence of procaine, noradrenaline activates dihydropyridine‐sensitive calcium channels through α‐adrenoceptors. 5 These findings indicate that, in the rat vas deferens, noradrenaline induces contraction by releasing intracellularly stored Ca2+. The effects of procaine appear to be due to its ability to block the release of Ca2+ from intracellular stores. Furthermore, the simultaneous increase in 45Ca influx and inhibition of inositol‐1,4,5‐trisphosphate formation in tissues treated with procaine plus noradrenaline indicates that Ca2+ influx is independent of inositol‐1,4,5‐trisphosphate formation.

Inositol 1,4,5‐trisphosphate and inositol 1,3,4,5‐tetrakisphosphate binding sites in smooth muscle

1 We have previously demonstrated that activation of M3 muscarinic receptors increases inositol 1,4,5‐trisphosphate (InsP3) and inositol 1,3,4,5‐tetrakisphosphate (InsP4) accumulation in colonic smooth muscle. 2 In the present study, we demonstrate the existence of InsP3 and InsP4 binding sites in colonic circular smooth muscle by use of radioligand binding methods. Both [3H]‐InsP3 and [3H]‐InsP4 bound rapidly and reversibly to a single class of saturable sites in detergent‐solubilized colonic membranes with affinities of 5.04 ± 1.03 nm and 3.41 ± 0.78 nm, respectively. The density of [3H]‐InsP3 binding sites was 335.3 ± 19.3 fmol mg−1 protein which was approximately 2.5 fold greater than that of [3H]‐InsP4 sites (127.3 ± 9.1 fmol mg−1 protein). 3 The two high affinity inositol phosphate binding sites exhibited markedly different pH optima for binding of each radioligand. At pH 9.0, specific [3H]‐InsP3 binding was maximal, whereas [3H]‐InsP4 binding was only 10% that of [3H]‐InsP3. Conversely, at pH 5.0, [3H]‐InsP4 binding was maximal, while [3H]‐InsP3 binding was reduced to 15% of its binding at pH 9.0. 4 InsP3 was about 20 fold less potent (K1 = 50.7 ± 8.3 nm) than InsP4 in competing for [3H]‐InsP4 binding sites and could compete for only 60% of [3H]‐InsP4 specific binding. InsP4 was also capable of high affinity competition with [3H]‐InsP3 binding (K1 = 103.5 ± 1.5 nm), and could compete for 100% of [3H]‐InsP3 specific binding. 5 [3H]‐InsP3 binding in subcellular fractions separated by discontinuous sucrose density gradients followed NADPH‐cytochrome c reductase activity, suggesting an intracellular localization for the majority of InsP3 receptors in this tissue, whereas [3H]‐InsP4 binding appeared to be equally distributed between plasma membrane and intracellular membrane populations. 6 These results suggest the existence of distinct and specific InsP3 and InsP4 binding sites which may represent the physiological receptors for these second messengers; differences in the subcellular distribution of these receptors may contribute to differences in their putative physiological roles.

Effects of potassium and norepinephrine on calcium influx in guinea-pig vas deferens

The effects of potassium (K+) and adrenoceptor agonists on 45Ca influx and contraction of isolated vas deferens from reserpine-treated guinea-pigs has been investigated. K+ (18-84 mM) increased 45Ca influx with the maximum influx occurring at 50 mM K+. The rate of influx upon exposure to K+ was highest in the first 2 min and decreased thereafter. In contrast to K+, norepinephrine, methoxamine and xylazine did not increase the rate of 45Ca influx. Norepinephrine and methoxamine, but not xylazine, inhibited 45Ca influx induced by high K+. The inhibitory effect of norepinephrine was also present in denervated tissues. Prazosin, but not yohimbine or propranolol, blocked the inhibitory effect of norepinephrine. Potassium-induced contractions were significantly potentiated in the presence of norepinephrine, especially at low calcium concentrations. We conclude that K+ contracts the guinea-pig vas deferens by increasing the influx of calcium whereas stimulation of alpha 1-adrenoceptors prevents 45Ca influx elicited by high potassium.

Factors involved in the generation of tension during contraction to high potassium in the rat vas deferens.

A large number of studies indicate that K(+)-induced contractions of smooth muscle depend on extracellular calcium. If these contractions depend exclusively on extracellular calcium then contractile responses to 140 mM K+, which are larger than the response to 35 mM K+, should be associated with a larger influx of 45Ca. This is not the case in the vas deferens from reserpine pretreated rats. During a 2 min interval, 45Ca influx induced by 140 mM K+ was identical to that produced by 35 mM K+. This suggests that a second mechanism may be involved in responses to high K+. Indeed, 140 mM K+ caused an approximately 300% increase above control in the formation of inositol trisphosphate (IP3) in tissues prelabelled with 3H-myoinositol whereas 35 mM K+ did not increase IP3. IP3 is thought to cause the release of calcium from internal stores which is consistent with our finding of an increase in 45Ca efflux into calcium-free medium from tissues prelabelled with 45Ca and stimulated with 140 mM K+. Stimulation with 35 mM K+ did not influence 45Ca efflux. We conclude that in the rat vas deferens high K+ promotes tension development by smooth muscle by a dual mechanism: influx of extracellular calcium and release of calcium from internal stores via an IP3 mechanism.