Muscarinic cholinergic stimulation of phosphatidylinositol turnover in the longitudinal smooth muscle of guinea-pig ileum.

1. The metabolism of phosphatidylinositol and phosphatidate was investigated in fragments of longitudinal smooth muscle from guinea-pig ileum incubated with cholinergic and anticholinergic drugs. 2. Incorporation of Pi into these lipids was enhanced by acetylcholine and carbamoylcholine. 3. The receptor responsible for triggering this response was of the muscarinic type, since (a) the response was also produced by the muscarinic agonists acetyl-beta-methylcholine, carbamoyl-beta-methylcholine and pilocarpine, and (b) the response was prevented by atropine and prophylbenzilylcholine mustard, but not by tubocurarine. 4. Increased phosphatidylinositol labellin was clearly observed within 5 min in tissue treated with a high concentration of carbamoylcholine. 5. Halfmaximal stimulation of phosphatidylinositol labelling occurred at approx. 10 muM-muM-carbamoylcholine. 6. Incubation of muscle fragments with carbamoylcholine provoked a decrease in phosphatidylinositol concentration, as would be expected if phosphatidyl-inositol breakdown is the reaction controlled by agonists. 7. This information all appears consistent with the proposal that phosphatidylinositol breakdown may be a reaction intrinsic to the mechanisms of muscarinic cholinergic receptor systems.     

Muscarinic-receptor stimulation enhances polyphosphoinositide breakdown in guinea-pig ileum smooth muscle.

Muscarinic-receptor stimulation by 0.1 mM-carbachol in longitudinal muscle of the guinea-pig ileum increases the incorporation of [3H]inositol into inositol-containing phospholipid. This effect was blocked by 16 microM-atropine. After 60 min incubation, carbachol increased the accumulation of total inositol phosphates 20-fold in the presence of 10 mM-Li+. Less than 20% of the total inositol phosphate corresponded to inositol 1-phosphate by ion-exchange chromatography, whereas of the remainder about two-thirds corresponded to inositol bisphosphate and one third to inositol trisphosphate. It is concluded that stimulation of muscarinic receptors in guinea-pig ileum enhances breakdown of polyphosphoinositides, suggesting that this may be a primary event associated with Ca2+ mobilization in the guinea-pig ileum.     

Dinitrofluorobenzene-induced rigor in ileal longitudinal smooth muscle of the guinea-pig.

1. The administration of 2,4-dinitrofluorobenzene (DFB) (0.1-1 mM) to the ileal longitudinal muscle produced contractions within seconds of its administration. 2. A component of the first 2 min duration of the phasic phase of 1 or 0.5 mM DFB contraction and the first phase of 0.35 or 0.1 mM DFB contraction was inhibited by Ca2+ antagonists, 1 x 10(-6) M D-600. 3. The DFB contraction resistant to D-600 began to develop when the tissue ATP concentration rapidly reduced. 4. The DFB contraction in ileum consists of two components; an initial fast contraction which is sensitive to Ca2+ antagonists, and a late contraction referred to as a rigor which is resistant to it.     

Nexuses between the smooth muscle cells of the guinea-pig ileum

The circular musculature of the guinea-pig ileum has been studied by freeze-fracture to analyze quantitatively the gap junctions (nexuses) between its smooth muscle cells. The average cell surface area and cell volume are 5,074 micron 2 and 3,260 micron 3. The packing density of nexuses is 48/1,000 micron 2 of cell surface or approximately 244/muscle cell. Nexuses range in area from less than 0.1 to approximately 1.5 micron 2 and they occupy 0.212% of the cell surface. The average packing density of intramembrane particles or pits in nexuses is approximately 7,200/micron 2 of nexal surface, indicating that there may be approximately 77,000 intercellular channels in the full complement of nexuses of one muscle cell.     

Short-term desensitization of phosphatidylinositol turnover via muscarinic acetylcholine receptors and histamine H1-receptors in smooth muscle

Smooth muscle of guinea-pig taenia caecum was desensitized by treatment with 10(-4)M carbachol or 10(-4)M histamine for 30 min in Ca-free solution containing 2mM EGTA. Phosphatidylinositol turnover stimulated by carbachol was not reduced by desensitization with either carbachol or histamine, while the turnover stimulated by histamine was reduced by desensitization with histamine, but not with carbachol. These results are consistent with our previous report (1) that heterologous desensitization induced by carbachol occurs at intracellular Ca stores and homologous desensitization by histamine occurs at H1 receptors.     

Interactions of neurogenic responses of longitudinal and circular muscle in the guinea-pig ileum

The relationship between neurogenic responses of longitudinal and circular muscle was studied by measuring contractions and EMG or nonadrenergic, non-cholinergic (NANC) relaxations and NANC inhibitory junction potentials in different preparations of the guinea-pig ileum. NANC relaxation of longitudinal muscle was observed also without any preceding or concomitant circular muscle contraction ruling out the possibility that the latter might be the cause of the NANC relaxation. Circular muscle twitches or powerful contractions were absent if there was no preceding neurogenic or myogenic excitation of longitudinal muscle; in preparations with myenteric plexus-longitudinal muscle layers removed only small residual responses were seen although still under neurogenic influences. Thus excitation of longitudinal muscle seemed a prerequisite for synchronized and powerful contractions of circular muscle to occur. Cholinergic contraction and NANC relaxation of longitudinal muscle evoked by field stimulation were partly inhibited if the submucous plexus was also present suggesting the involvement of a more complex neuronal circuitry in these responses.     

Effect of papaverine on the longitudinal and circular smooth muscle of the guinea-pig caecum.

Electrical and mechanical activity of smooth muscle of the guinea-pig caecum was recorded by means of the sucrose-gap technique. The responses of longitudinal and circular smooth muscle to acetycholine were differently affected by changes of extracellular calcium concentration (0.8, 2.5 and 7.5 mM). The contractions of both preparations were depressed at high Ca++ concentrations, whereas at low Ca++ concentrations only contractions of the circular smooth muscle were augmented. The stimulatory effect of acetylcholine was decreased by papaverine in both preparations at all three concentrations of Ca++. This inhibition was the greater the lower the concentration of extracellular Ca++ and this process was more pronounced in the circular muscle. The ability of papaverine to counteract the effect of lowered concentrations of extracellular Ca++ on membrane excitability may well explain its inhibitory effect upon intestinal smooth muscle.     

Iodoacetic acid-induced rigor in ileal longitudinal smooth muscle of guinea-pig

1. Ileal tensions to iodoacetic acid (IAA) develop when tissue ATP concentration falls below approximately 60-55% of the control. 2. As the IAA concentration is increased (0.1-10 mM), the ATP concentrations decrease rapidly, and both the time of the onset and the duration of the contraction shorten. 3. In the presence of lactate, IAA failed to decrease the tissue ATP concentration and did not develop tension. 4. The contraction to IAA developed in the presence of Ca2+ antagonist, D-600 or in Ca2(+)-free solution, however, onset time was prolonged. 5. These results suggest that the contraction to IAA is referred to as 'rigor' because it increases with decreasing tissue ATP concentration in ileum.     

Presynaptic and postsynaptic effects of mercuric ions on guinea-pig ileum longitudinal muscle strip preparation

The toxic effect of mercuric ions on intestinal cholinergic neurotransmission was investigated in vitro. Hg²⁺ inhibited the evoked release and enhanced the resting release of ACh. Smooth muscle contraction was irreversibly inhibited by Hg²⁺ in a concentration-dependent manner, and Na₂EDTA did not antagonize this effect. We also investigated if Hg²⁺ enters the nerve terminal through Ca²⁺-channels, or Na⁺-channels, or both. The effects of mercuric ions obtained in our study were completely abolished by the combined administration of TTX and Co²⁺. It is suggested that the site of the action of mercuric ions is intracellular. We concluded that Hg²⁺ may interfere with cholinergic transmission by blocking [Ca²⁺]_o-dependent release of ACh and by enhancing [Ca²⁺]_o-independent resting release of ACh. The effect of Hg²⁺ was not only presynaptic since it also inhibited the effect of ACh on smooth muscle.     

Monoamine and diamine oxidative deamination in the longitudinal smooth muscle of guinea pig ileum

The longitudinal smooth muscle of guinea pig ileum contains three different types of oxidative deaminating enzymes: monoamine oxidase types A and B, diamine oxidase and a soluble clorgyline-deprenyl-resistant benzylamine oxidase. These enzymes have different subcellular locations. The longitudinal smooth muscle of guinea pig ileum oxidatively deaminates beta-phenylethylamine at a much higher rate than benzylamine. beta-Phenylethylamine is a good substrate for monoamine oxidase type B but also for the soluble clorgyline-deprenyl-resistant benzylamine oxidase. On the other hand, benzylamine is oxidised by mitochondrial monoamine oxidase, by the clorgyline-deprenyl-resistant enzyme and by diamine oxidase.     

Interaction between histamine and vagal stimulation on tracheal smooth muscle in dogs

We examined the interaction between histamine and vagal efferent activity on airway smooth muscle reactivity in 11 anesthetized vagotomized dogs using an isolated closed segment of the intrathoracic trachea filled with Tyrode solution under an isovolumetric condition. Intratracheal pressure change was measured as an index of tracheal smooth muscle tone. The administration into the tracheal segment of histamine (0.1 or 1.0 mg/ml) in six dogs and methacholine chloride (0.001 or 0.01 mg/ml) in the other five dogs elevated intratracheal pressure by about 5 cmH2O. The electrical stimulation of the peripheral ends of both of the cut cervical vagus nerves in the presence of histamine produced significantly greater responses than the additive responses of these two stimuli applied individually (two-way analysis of variance, P less than 0.025). However, the combined effects of vagal stimulation and methacholine were not significantly different from the additive responses of these two stimuli applied individually. The average values of intratracheal pressure elevated by the combined effects of vagal stimulation and histamine were significantly higher than those obtained by the combination of vagal stimulation and methacholine (two-way analysis of variance, P less than 0.01). This suggests that histamine potentiates tracheal smooth muscle reactivity to electrical vagal stimulation, which may contribute to the hyperreactivity observed in patients with asthma.     

Protein phosphatase composition in the smooth muscle of guinea-pig ileum studied with okadaic acid and inhibitor 2.

Using okadaic acid, a potent inhibitor of type 2A and type 1 protein phosphatases, and inhibitor 2, an intrinsic inhibitory factor of type 1 phosphatase, we characterized the phosphorylated myosin light-chain (PMLC) phosphatase activity in the smooth-muscle extracts of guinea-pig ileum. In the intact fibres the control activity was 254 +/- 13 nmol of Pi/min per g wet wt. (n = 15) against 32P-labelled PMLC (4 microM) from chicken gizzard. The following phosphatase fractions were identified: an inhibitor-2-sensitive (type 1) fraction (fractional activity = 35%), a Mg2+-dependent and okadaic acid-insensitive (type 2C) fraction (17%), and two type 2A-like fractions that had different susceptibility to okadaic acid. The type 2A-like fraction with lower affinity to okadaic acid accounted for 30% of the control activity. After the cell membrane was permeabilized by Triton X-100, more than 60% of this fraction remained and accounted for about 90% of the total activity, whereas the other fractions were nearly abolished. The type 2A-like fraction may be bound to some intracellular structure such as contractile proteins.     

Hydrolysis of biological materials for histamine determination on the isolated guinea-pig ileum.

Conditions of hydrolysis of biological material for determinations of histamine (Hi) using the guinea-pig ileum method were investigated. Overheated vapour has been applied at varying intervals and it was found that optimal conditions for tissue He liberation are obtained after 2 hour hydrolysis in autoclave. Results were compared with the orginal Code's method.     

Differential effects of phenylmethanesulfonyl fluoride (PMSF) on carbachol and potassium stimulated phosphoinositide turnover and contraction in longitudinal smooth muscle of guinea pig ileum

Phenylmethanesulfonyl fluoride (PMSF) (2 mM), a putative inhibitor of phosphatidylinositol-specific phospholipase C, almost completely inhibited carbachol-stimulated inositol incorporation into phosphatidylinositol (PI) of longitudinal smooth muscle of guinea pig ileum, while it had no effect on potassium-stimulated inositol incorporation. This suggests that the two stimuli may affect phosphoinositide turnover by different mechanisms, distinguishable by PMSF. In contrast to its specific inhibition of carbachol-stimulated phosphoinositide turnover, PMSF produced a transient inhibition of contraction by both carbachol and potassium. The non-selective effect of PMSF on contraction suggests that it is not the result of its inhibitory effect on phosphoinositide breakdown. PMSF (2 mM) inhibited carbachol-stimulated inositol phosphate accumulation in the presence of Li+ by only 15%-19%, indicating that PMSF inhibition of phosphoinositide turnover was not due to its inhibition of phosphoinositide phosphodiesterase, but to one or more steps following phosphoinositide breakdown.     

Vascular vasopressin receptors mediate phosphatidylinositol turnover and calcium efflux in an established smooth muscle cell line

Vasopressin-induced phosphatidylinositol turnover and mobilization of intracellular Ca2+ was studied using an established smooth muscle cell line (A-10). The cells were subcloned to ensure a monoclonal cell population. The accumulation of inositol mono-, di-, and tris-phosphates (IP1, IP2, and IP3, respectively), and the mobilization of intracellular Ca2+ were dependent on the time of incubation and the concentration of arginine vasopressin (AVP). IP1, IP2, and IP3 were significantly elevated after 15 sec and remained elevated for up to 2 hr. The concentrations of AVP required for half-maximal stimulation of IP1, IP2, and IP3 formation were 2, 12, and 4 nM, respectively. LiCl was required to observe the accumulation of inositol phosphates in response to AVP. Significant 45Ca2+ efflux was observed within 15 sec after exposure to AVP. By employing the vasopressin receptor subtype selective antagonists [d(CH2)5Tyr(Me)AVP, V1; d(CH2)5D-Tyr(Et)VAVP,V1/V2; d(CH2) 5D-IleVAVP,V2] and agonists [AVP, V1/V2; dDAVP, V2; dVDAVP, V2], we found that the vasopressin-induced stimulation of phosphatidylinositol turnover and 45Ca2+ efflux were mediated by receptors of the vascular V1 subtype. Pertussis toxin pretreatment partially inhibited vasopressin-induced phosphatidylinositol turnover. These data demonstrate that activation of V1 receptors of vascular smooth muscle cells resulted in enhanced phosphatidylinositol turnover and mobilization of intracellular Ca2+.