Tetrodotoxin-resistant contractions induced by electrical stimulation of bladder muscle from man, rabbit and rat

Isolated detrusor preparations from man, rabbit and rat were suspended in an organ bath and isometric tension was recorded. The preparations were stimulated electrically in the presence of Bay K8644 and nifedipine before and after neuronal blockade with tetrodotoxin. Transmural electrical stimulation produced frequency-dependent contractions in all preparations. Bay K8644 significantly increased and nifedipine decreased these contractions. TTX effectively suppressed the response to electrical field stimulation in all species. When Bay K8644 was added to TTX blocked preparations, the responses to electrical stimulation were partly restored in bladder strips from man and rat. No increase in response was seen in the rabbit preparations. However, if the extracellular K+-concentration was increased to 10 mM (which per se did not affect the response) Bay K8644 significantly increased the contractions. All responses elicited by electrical stimulation in the presence of TTX were abolished by nifedipine. It is concluded that if the bladder smooth muscle is exposed to factors that can increase its sensitivity to contractile agents, this may result in uncontrolled (unstable) bladder contractions. Such contractions may use the 'normal' transmitter substances, but may be triggered at a lower stimulus intensity than normal. As a non-specific increase in membrane excitability seems to be associated with an influx of calcium through voltage-sensitive calcium channels, calcium antagonists, together with agents specifically blocking relevant transmitter substances, would offer an effective therapy against the unstable bladder.     

Different effects of neuropeptide Y on electrically induced contractions in the longitudinal and circular smooth muscle layers of the female rabbit urethra

The effects of neuropeptide Y (NPY) on preparations of isolated longitudinal and circular smooth muscle from rabbit urethra were studied. In both types of muscle, electrically induced contractions and relaxations could be abolished by tetrodotoxin, (TTX). In the longitudinal muscle preparations the contraction was slightly reduced by prazosin, but markedly reduced by scopolamine and NPY. The NPY effect was not influenced by pretreatment with rauwolscine. Pretreatment with NPY had no effect on contractions induced by noradrenaline (NA) or carbachol and the peptide did not relax preparations contracted by these agents. In circular muscle an initial, fast response, not sensitive to prazosin or scopolamine was occasionally observed following electrical stimulation. A slow contraction component was regularly seen; this response was abolished by prazosin. Neuropeptide Y did not influence any of these responses. The preparations were concentration-dependently contracted by NA, whereas carbachol had no effect. Pretreatment with NPY did not affect contractions induced by NA, nor did the peptide relax NA-contracted preparations. In neither longitudinal nor circular muscle strips did NPY affect the electrically induced TTX sensitive relaxation of NA-contracted preparations. The results suggest that in the rabbit urethra NPY reduces contractions in the longitudinal muscle layer by selectively inhibiting the release of acetylcholine from cholinergic nerves. Neuropeptide Y did not appear to have any significant postjunctional effects nor to interfere with the release, or effects of NA or other transmitter agents. The physiological importance of the urethral effects of NPY remains to be established.     

Relaxation mechanisms induced by stimulation of nerves and by nitric oxide in sheep urethral muscle.

Isolated transverse and longitudinally oriented preparations of sheep urethra precontracted with noradrenaline responded to electrical field stimulation (EFS) with stimulus-dependent non-adrenergic, non-cholinergic (NANC) relaxations. Exogenous nitric oxide (NO) (acidified NaNO2), S-nitroso-L-cysteine (NC), sodium nitroprusside (SNP), 8-Br-cGMP, dibutyryl-cAMP, forskolin and isoprenaline each relaxed precontracted transverse urethral preparations in a concentration-dependent manner in order of protency: NC > forskolin > isoprenaline = SNP > NO > 8-Br-cGMP = dibutyryl-cAMP. Longitudinally oriented preparations responded to NO and NC with concentration-dependent relaxation, no different from that observed in transverse strips. Methylene blue (MB) and oxyhaemoglobin (HbO2) each shifted the concentration-response curve for NO to the right without affecting EFS-induced relaxation. Similarly, concentration-dependent responses to NC were not affected by MB. The inhibition of relaxation to NO by MB was prevented by superoxide dismutase, suggesting the inhibition was caused by extracellular generation of superoxide anions. EFS-induced relaxation was accompanied by elevation of cGMP. However, for the same level of relaxation, exogenous NO and NC induced 15- and 23-times higher increases in cGMP values, respectively, than EFS. cAMP levels were not affected by EFS- or NO-induced relaxation, although a large increase accompanied relaxation induced by forskolin. Forskolin also increased cGMP content. Pretreatment with MB reduced basal levels of cGMP and inhibited both relaxation and rise in cGMP levels induced by NO. SNP-elicited relaxant responses, in the presence of MB, were accompanied by an accumulation of cGMP; cAMP levels were unaffected. MB reduced cGMP levels induced by NC, while the relaxant response was unchanged. In urethral preparations prelabelled with [3H]myoinositol, exposure to NA caused an accumulation of [3H]inositol phosphates, which was unaffected by pretreatment with 8-Br-cGMP or dibutyryl-cAMP. EFS failed to induce a relaxant response in excess [K+]o-contracted preparations, while relaxation with exogenous NO was unaffected. Ouabain abolished EFS-induced relaxation and reduced responses to NO. Neither TEA nor glibenclamide affected relaxation to either EFS or NO. Relaxation elicited by SNP was not accompanied by any change in cGMP or cAMP levels, and was unaffected by MB, HbO2, K+ channel blockers (TEA and glibenclamide), ouabain or high [K+]o solution. This suggested that relaxation was caused by a mechanism independent of NO generation. A dense network of NADPH diaphorase-positive fibres associated with both the circular and longitudinal smooth muscle layers of sheep urethra was found.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cholinergic and adrenergic neural control of smooth muscle function in the non-pregnant rat uterine cervix

The isolated circular smooth muscle of the uterine cervix from spayed rats was investigated in vitro. One group of animals was treated with oestrogen. Preparations from these rats showed no spontaneous contractile activity, but responded with contractions to electrical field stimulation. The contractions were blocked by approximately 85% with atropine, scopolamine and tetrodotoxin, but were unaffected by the adrenergic antagonists propranolol, phenoxybenzamine, and yohimbine. This neurogenic effect was potentiated by neostigmine. Noradrenaline inhibited the nerve-induced contractions and also lowered resting tension in a concentration-dependent way. The latter effect of noradrenaline was inhibited by propranolol. The indirect effects of noradrenaline on the electrically induced contractions were also inhibited by propranolol, but potentiated with phenoxybenzamine and, to a less extent, yohimbine. Nerve-induced relaxation was never observed. Acetylcholine contracted the smooth muscle by an effect that was counteracted by atropine. A second group of spayed rats was not given any steroid treatment. The cervix preparations from these animals showed spontaneous activity, which was uninfluenced by atropine, propranolol, phenoxybenzamine, yohimbine, and tetrodotoxin. Noradrenaline inhibited the spontaneous activity. This noradrenergic effect was counteracted by propranolol and potentiated by phenobenzamine. Acetylcholine increased the smooth-muscle tone by an atropine-sensitive action. The results suggest the presence of a cholinergic motor innervation on the non-pregnant rat uterine cervix. The cervix also receives an adrenergic innervation, which may act both post-junctionally (lowering resting tension and inhibiting spontaneous activity) and prejunctionally (modulating the cholinergic nerves) via beta-adrenergic as well as alpha-adrenergic receptors.     

Concentrations and contractile effects of substance P in the human ampullary-isthmic junction

From 20 women undergoing hysterectomy, strip preparations were isolated from the outer, longitudinal and the inner, circular smooth muscle layer of the ampullary-isthmic junction (AIJ), together with small arterial segments dissected as ring preparations from the root of the mesosalpinx. The specimens were mounted in organ baths and isometric tension was recorded. In addition, tissue concentrations of substance P (SP) in the ampulla, AIJ and utero-tubal junction were determined by radioimmunoassay. Tissue concentrations of SP expressed as pmol X g tissue-1 (wet weight, +/- SE) amounted to 3.09 +/- 1.40 in the utero-tubal junction, 1.08 +/- 0.299 in the AIJ and 0.742 +/- 0.299 in the ampulla. In strips of circular muscle, SP at concentrations of 10(-7) -3 X 10(-6) mol X l-1 elicited a combined phasic and tonic response and in longitudinal muscle a mainly tonic contraction was produced. In both tissues, contractions elicited by SP were rapidly abolished in calcium-free medium. Nifedipine abolished the phasic contraction elicited in circular muscle by SP while the tonic response was resistant. The contraction in longitudinal muscle was reduced by 20-30%. Vasoactive intestinal polypeptide (VIP) decreased tension in preparations contracted by SP, prostaglandin F2 alpha and K+-depolarization (124 mmol X l(-1). In unstimulated oviductal arterial preparations, SP had no effect, while the peptide induced a transient relaxation of noradrenaline contracted preparations, and slightly decreased tension of K+-depolarized vessels. The results suggest that SP may be involved in the control of motility of the human AIJ.     

Effects of some peptides on isolated human penile erectile tissue and cavernous artery

Contractant and relaxant effects of four peptides known to occur in nerves innervating human penile vessels and erectile tissue, namely substance P (SP), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY) and somatostatin, were studied in isolated preparations from the corpus cavernosum (CC), corpus spongiosum (CS) and cavernous artery (Acc). In addition, the actions of another peptide, arginine vasopressin (AVP), were investigated. In erectile tissue proper, SP induced concentration-dependent contractions. No effect of this peptide was observed in Acc segments. CC and CS preparations contracted by noradrenaline (NA) were relaxed by 30-40%; the effect in NA-contracted Acc preparations was inconsistent. AVP had a potent contractant effect in preparations from all the tissues studied, the effect being most conspicuous in CS strips. VIP was without contractant actions in any of the preparations. NA-contracted preparations were relaxed by VIP, and electrically induced contractions inhibited. The inhibitory effect was particularly marked in electrically stimulated CC and CS preparations. NPY had no effects; somatostatin contracted Acc segments, and in high concentrations CC and CS strips. It is concluded that among the peptides studied only VIP has effects compatible with a role as a neurotransmitter in penile erection.     

Relaxant responses to transmural stimulation and nicotine of dog and monkey cerebral arteries

In helical strips of dog and monkey cerebral arteries contracted with prostaglandin F2 alpha, transmural stimulation and nicotine produced relaxations that were abolished by tetrodotoxin and hexamethonium, respectively. These responses were attenuated by quinidine, whereas relaxations of dog coronary arteries to transmural stimulation and isoproterenol were unaffected. Treatment with vasoactive intestinal polypeptide (VIP) and substance P (SP) abolished the relaxant response of cerebral arteries to repeated applications of VIP and SP, respectively; however, after VIP or SP, a normal relaxant response to transmural stimulation or nicotine was produced. Aminophylline suppressed relaxations induced by ATP but not by nerve stimulation. VIP, SP, and adenosine 5'-triphosphate (ATP) relaxed dog cerebral arteries; the responses were unaffected by quinidine. However, only VIP and ATP relaxed monkey cerebral arteries, and SP contracted the arteries. Acetylcholine contracted monkey arteries, in which transmural stimulation produced a relaxation. It may be concluded that nerves innervating the cerebrospinal wall are stimulated electrically and chemically by nicotine, resulting in the arterial relaxation. However, a vasodilator transmitter was not identified. Quinidine appears to selectively antagonize the action of the transmitters on cerebroarterial smooth muscle.     

Localization of Adrenergic Receptors in Guinea Pig Ileum and Rabbit Jejunum to Cholinergic Neurons and to Smooth Muscle Cells

The localization of adrenergic receptors mediating a relaxing action was investigated in innervated and denervated longituclinal muscle strips from guinea pig ileum and rabbit jejunum. Denervated preparations were contracted by drugs that had a direct effect on smooth muscle cells, such as acetylcholine and histamine, but not by stimuli acting on cholinergic neurons, such as electrical field stimulation or nicotine. After blockade of β-adrenoceptors, norepinephrine relaxed the innervated guinea pig ileum contracted by electrical field stimulation, by stimulating α-adrenoceptors. Norepinephrine in low concentrations did not relax denervated preparations contracted by agents acting directly on smooth muscle. In high concentrations, it relaxed denervated preparations by a nonadrenergic mechanism, resistant to α- and/or β-receptor blockade, but which was also activated by l-(3,4-dihydroxyphenyl) ethanol. Phenylephrine only had a weak agonistic effect on the electrically stimulated innervated preparation and did not relax the denervated one. The denervated rabbit intestine contracted by acetylcholine was relaxed by norepinephrine and phenylephrine by stimulation of α-adrenoceptors. In the innervated preparations both drugs were more effective in inhibiting contractions induced by electrical field stimulation or eserine than those induced by exogenous acetylcholine. Both the denervated guinea pig and rabbit intestine were relaxed by stimulation of β-adreno-ceptors. It is suggested that in the guinea pig ileum α-adrenoceptors mediating relaxation are located only in cholinergic neurons, whereas in rabbit jejunum they are located both in these neurons and in the smooth muscle cells. Beta-adrenoceptors are located in the smooth muscle cells of both organs.     

Regulation of voltage-dependent excitatory responses to alpha,beta-methylene ATP, ATP and non-adrenergic nerve stimulation by dihydropyridines in the guinea-pig vas deferens

Simultaneous recordings of mechanical and intracellular electrical activity were obtained from the guinea-pig vas deferens, where nerve stimulation, ATP and the stable nucleotide analogue alpha,beta-methylene ATP elicited excitatory responses. Excitatory junction potentials and action potentials were elicited by low-frequency (trains of pulses, generally less than or equal to 2 Hz) field stimulation. alpha,beta-Methylene ATP and ATP elicited only concentration-dependent depolarizations at low concentrations, while higher concentrations elicited a superimposed action potential discharge which was accompanied by mechanical contraction. The voltage threshold at which action potential discharge was initiated by these three stimuli was about -45 mV (resting membrane potential averaged -66 mV). Action potential discharges and contractile responses were antagonized by nifedipine and augmented by Bay K 8644 at concentrations (1 and 0.5 microM, respectively) which exhibited only small effects on either excitatory junction potential amplitudes or nucleotide-induced depolarizations. Bay K 8644 enhanced and nifedipine antagonized the repolarization (rectification) phase of action potential discharge elicited by nerve stimulation and drugs; after-hyperpolarizations were prominent in the presence of Bay K 8644 (0.1-5 microM). Excitatory junction potentials were antagonized after exposure to alpha,beta-methylene ATP. This antagonistic effect of alpha,beta-methylene ATP was also observed following depolarizations elicited in the absence and presence of nifedipine (1 microM). Noradrenaline was approximately 50-100 times less potent than alpha,beta-methylene ATP in eliciting action potential discharge and contraction. It was only when a high concentration of noradrenaline was used (about 60-100 microM) that the noradrenaline-induced depolarization attained the voltage threshold for action potential initiation. These results illustrate the similarity of the electrical components which underlie excitation by nerve stimulation and adenine nucleotides in the vas deferens, and demonstrate the ability of dihydropyridines to regulate voltage-dependent events associated with both the generation and inactivation of muscle action potentials. These are probably voltage-dependent calcium currents and calcium-activated potassium currents, respectively. Neither excitatory junction potentials nor the mechanism of desensitization of the ATP purinoceptor by alpha,beta-methylene ATP involve voltage-dependent calcium channels.     

Effects of chloroquine on smooth muscle contracted with noradrenaline or high-potassium solutions in the rat thoracic aorta.

The effects of chloroquine on the smooth muscle of isolated rat aortic segments were investigated in preparations contracted with either noradrenaline or high-potassium. At rest, chloroquine (up to 10(-4) M) produced no mechanical response, while noradrenaline (10(-6) M) produced a sustained contraction. In the presence of 10(-4) M chloroquine, however, the amplitude of contractions produced by noradrenaline was attenuated by about 70%, with no alteration of the resting tension. In preparations contracted either with noradrenaline or with high-K solutions, chloroquine produced a concentration-dependent relaxation. The tension decreased below resting level as a result of the co-application of these stimulants. The relaxing actions of chloroquine were not altered by methylene blue (an inhibitor of guanylate cyclase), suggesting that the cyclic GMP-related mechanism was not involved. The ratio of the amplitude of chloroquine-induced relaxation was similar in contractions produced by different concentrations of potassium ions, suggesting that chloroquine did not cause relaxation as a result of membrane hyperpolarization. These results suggest that the inhibition of aortic smooth muscle contraction caused by chloroquine is different to that produced by endothelium-derived vasodilating factors. It is possible that the inhibition of aortic smooth muscle contraction by chloroquine involves modulation of the contractile systems and of their regulatory proteins.     

Contractile effects of endothelin-1 and localization of endothelin binding sites in rabbit lower urinary tract smooth muscle

In isolated rabbit bladder and urethral smooth muscle, endothelin-1 caused concentration-related, slowly developing contractions that were difficult to wash out. Relative to contractions induced by K+ (124 mM), contractions in bladder preparations reached a higher amplitude than in urethral preparations. There was a marked tachyphylaxis to the effects of the peptide. The endothelin-1-induced contractions were not significantly affected by phentolamine or indomethacin in the urethra, or by scopolamine or indomethacin in the bladder. Incubation for 30 min in a Ca2(+)-free solution abolished the endothelin-1-induced contractions. Nifedipine did not affect the actions of endothelin-1 in the urethra but had a marked inhibitory action on its effects in the bladder. In the presence of endothelin-1, Ca2(+)-induced contractions were significantly blocked by nifedipine in the bladder but not in the urethra. Urethral preparations at resting tension responded to electrical stimulation by tetrodotoxin-sensitive, frequency-dependent contractions sensitive to alpha-adrenoceptor blockade. Pretreatment with endothelin-1 (10(-9) M) produced a significant increase in the nerve-induced contractions but had no significant effect on contractions induced by exogenous noradrenaline. Endothelin-1 did not affect spontaneous or stimulation-induced efflux of 3H-labelled noradrenaline in urethral smooth muscle. Preparations contracted by endothelin-1 were frequency-dependently relaxed by electrical stimulation. The peptide had no significant effect on the responses induced by electrical stimulation in the bladder preparations. In both bladder and urethra, [125]endothelin-1 binding sites were found mainly in the outer longitudinal muscle layer, in vessels and in the submucosa. The highest density of binding sites appeared to be in vessels and the outer muscle layer in both types of muscle. The results suggest that in the rabbit both bladder and urethral smooth muscle contain binding sites for endothelin. The peptide has contractant effects dependent on extracellular calcium in both types of tissue, but voltage-operated calcium channels seem to involved in activation only of bladder smooth muscle. The functional importance of endothelin-1 in the rabbit lower urinary tract remains to be elucidated.     

Functional responses of different muscle types of the female rat urethra in vitro

The influence of muscle type on functional responses of the female rat urethra was investigated using morphological and functional in-vitro techniques. The urethral submucosa was found to contain longitudinally or obliquely oriented smooth muscle cells. The muscularis layer is composed of circularly oriented muscle cells. Near the bladder orifice smooth muscle fibres dominate, but in the mid-urethra the vast majority is circularly oriented striated muscle cells. Circular preparations responded to electrical field stimulation in vitro with a rapid contraction. Stimulation with single impulses resulted in a twitch response; frequencies exceeding 5-10 Hz induced a summation and tetanus. The response was unaffected by phenoxybenzamine, propranolol and scopolamine and had a low sensitivity to calcium-free solution but was sensitive to suxamethonium and tetrodotoxin. Using longer impulse trains stimulation evoked also a slow contraction, sensitive to calcium-free solution. In longitudinal preparations stimulation induced a relaxation followed by a contraction, responses much smaller than those seen in the circular preparations. Both preparations relaxed on addition of calcitonin gene-related peptide or capsaicin. The relaxation to calcitonin gene-related peptide was larger than that to capsaicin in longitudinal preparations but equally large in the circular ones. Substance P and 5-hydroxytryptamine contracted both preparations. The longitudinal urethra showed a larger contraction to 5-hydroxytryptamine than to substance P, whereas both substances induced similar responses in the circular preparations. The study shows a similar muscle arrangement in the female rat urethra as described in humans and further points to a functional differentiation between the different types of muscle.     

Effect of electrical field stimulation on muscle strips from chicken crop

The effect of electrical field stimulation (EFS) on smooth muscle strips from the chicken crop was investigated. The EFS (5 Hz, 2 ms, 400 mA; 10 Hz, 2 ms, 400 mA; 5 Hz, 2 ms, 500 mA; 10 Hz, 2 ms, 500 mA) produced contractions in all preparations. Atropine (10(-5) mol l-1) diminished muscle contraction in response to EFS. Tetrodotoxin (5 X 10(-6) mol l-1) fully suppressed the contractile responses to EFS. A serotonin antagonist (methysergide, 10(-4) mol l-1) did not alter contractions elicited by EFS. Propranolol (10(-5) mol l-1) led to an increase of contractions induced by EFS. Noradrenaline (10(-6)-10(-4) mol l-1) reduced acetylcholine-induced (1.1 X 10(-6) mol l-1) contraction of the crop smooth muscle strips. Propranolol (10(-5) mol l-1) abolished noradrenaline-induced relaxation almost fully. Tetrodotoxin (5 X 10(-6) mol l-1) did not change the relaxing effect of noradrenaline. These results suggested that the responses of crop smooth muscle to EFS involve excitatory cholinergic and non-cholinergic (probably peptidergic) as well as inhibitory adrenergic nerves.     

Endothelial mediated enhancement of noradrenaline induced vasoconstriction in normal and varicose veins

The role of the endothelium to influence smooth muscle contraction in normal and varicose veins was investigated in vitro using saphenous vein preparations obtained at vascular surgery. Paired control and endothelium-denuded rings were tested simultaneously and contracted with noradrenaline (10(-9)-10(-4) M). Identical experiments were also performed on sheep femoral veins and arteries. Noradrenaline induced maximal tension was 30% lower in varicose compared to normal veins. In normal veins removal of endothelium significantly reduced the maximal response to noradrenaline by 40% whereas in varicose veins no significant reduction could be seen. In the sheep femoral vein removal of the endothelium also resulted in decrease of noradrenaline-induced contraction. It can be concluded that in the human saphenous vein the endothelium has a contraction facilitating effect in response to stimulation with noradrenaline. In varicose veins the endothelial-mediated enhancement of noradrenaline-induced vasoconstriction is reduced probably because of endothelial damage. This observation may be of importance in the pathogenesis of varicose veins.     

Estrogen and the relaxant effect of intramural noradrenaline on calcium induced contractures in depolarized rat uterus

The influence of intramural noradrenaline on calcium induced contractures was studied in isolated preparations of rat uterus. The depolarized (127 mM KCl) myometrium of oophorectomized rats responded with contraction followed by a transient relaxation when exposed to 3 mM calcium. The threshold concentration of calcium, where the transient relaxation began to appear, was between 0.25 and 0.5 mM. Blockade of the beta-adrenoceptors with propranolol or noradrenaline depletion with reserpine completely removed the transient relaxation, indicating that the latter was due to release of intramural noradrenaline. Estrogen treatment abolished the relaxant effect of intramural noradrenaline, whereas progesterone was ineffective in this respect. Preparations from rats in natural estrus responded like estrogenized tissues, and diestrus preparations behaved as uteri of oophorectomized rats without estrogen treatment.     

Inhibitory effects of D2 agonists by striatal injection on excessive release of dopamine and hyperactivity induced by Bay K 8644 in rats

We investigated by means of behavioral and neurochemical studies the effects of either D(1) or D(2) agonist on excessive dopamine release and hyperactivity induced by the microinjection of Bay K 8644, and an L-type Ca(2+) channel stimulant, into the rat caudate putamen under a novel environmental condition. Hyperactivity (locomotor activity and rearing counts) and significant increases in extracellular dopamine levels induced by Bay K 8644 were concomitantly observed. D(1) agonist, SKF81297, administered into the caudate putamen did not block Bay K 8644-induced hyperactivity measured by monitoring both animal activity and increases in extracellular dopamine levels detected by microdialysis. Pretreatment with the D(2) agonists, bromocriptine, talipexole and pramipexole, into the caudate putamen significantly blocked Bay K 8644-induced hyperactivity for 45 min after Bay K 8644 administration, although the single administration of these agonists significantly potentiated locomotor activity and rearing behavior. Furthermore, these agonists significantly suppressed Bay K 8644-induced extracellular dopamine levels. Our results indicate that these D(2) agonists (1) act on postsynaptic neuronal D(2) receptors under conditions of normal or low dopamine release in the caudate putamen, and (2) act on presynaptic D(2) receptors (autoreceptors) when excessive levels of dopamine are released or hyperdopamine neuronal activity is induced. Consequently, the effect of D(2) agonists in the clinical treatment of Parkinson's disease may be due to stimulation of postsynaptic D(2) receptors rather than presynaptic autoreceptors.     

Inhibition of adrenergic neurotransmission in isolated veins of the dog by potassium ions.

1. In the intact organism, an increase in K+ concentration decreases the reactivity of blood vessels to sympathetic stimulation. The present experiments were designed to determine whether or not K+ interferes with adrenergic neurotransmission. 2. Helical strips cut from dogs' saphenous veins were incubated (4 hr) in Krebs-Ringer solution containing [7-3H]norepinephrine (5 times 10(-8) g/ml). The preparations were mounted for superfusion and isometric tension recording; the superfusate was collected for estimation of total radioactivity and for chromatographic separation of 3H-labelled norepinephrine and metabolites. 3. Supramaximal electric stimulation (5 Hz, 9 V, 2 msec) increased the tension and the [3H]norepinephrine efflux. Increasing the K+ concentration from 5-9 to 1, 15, and 20 m-equiv/l. caused a progressive depression of these contractions and diminished the total 3H efflux in proportion to the relaxation; the decrease in 3H efflux reflected a decrease in intact [3H]norepinephrine. The same increase in K+ concentration did not alter basal tension or basal 3H efflux. 4. Addition of tyramine (4 times 10(-6) g/ml. min) to the superfusate augmented both the tension and the efflux, but these actions were not depresesd by increasing the K+ concentration. 5. Cocaine, phentolamine, and phenoxybenzamine did not prevent the depression by K+ of the response to electric stimulation. 6. These experiments show that K+ causes relaxation of venous smooth muscle constricted by sympathetic stimulation and does so by inhibiting the release of norepinephrine from nerve endings. By contrast, K+ does not inhibit norepinephrine release in response to tyramine. 7. During submaximal electric stimulation (5 Hz, 1-8--3 V, 2 msec), increasing the K+ concentration from 5-9 to 10 and 15 m-equiv/l. potentiated the contractions and increased the [3H]norepinephrine efflux; at 20 m-equil/l, K+ caused transient increases in tension and 3H efflux followed by relaxation and decreased norepinephrine release. After addition of cocaine (10(-5) g/ml. min), K+ only caused relaxation and decrease in 3H efflux, showing that, in addition to inhibition of norepinephrine release, K+ also inhibits the reuptake process. 8. In higher concentrations (40 m-equil/l.), K+ caused both a liberation of norepinephrine and a direct activation of the smooth muscle cells.     

The Role of Intramural Noradrenaline in the Potassium Induced Contracture of Non-Estrogenized Smooth Muscle

The influence of intramural noradrenaline on potassium induced contractures was studied in isolated preparations of the uterus and the portal vein of the rat. Uterine strips of oophorectomized rats responded with contraction followed by transient relaxation when immersed in a medium containing 127 mM potassium chloride. Reserpinization or blockade of β-adrenoceptors with propranolol greatly diminished the transient relaxation. In the isolated portal vein, both noradrenaline depletion with reserpine and α-adrenoceptor blockade (phentolamine) reduced the active tension produced in response to the isotonic potassium solution. These results suggest that intramural noradrenaline plays a significant role in the development of contractures evoked by potassium in the uterus and the portal vein of the rat. In the uterus, the released noradrenaline counteracts the development of active tension, whereas in the portal vein, noradrenaline has a contractile effect which is added to that of potassium. Estrogen treatment reduced (portal vein) or abolished (rat uterus) the contractile effects of intramural noradrenaline. The adrenolytic effect of estrogen is possibly due to reduction in the release of noradrenaline from the sympathetic nerve-endings.     

The relation between stimulus frequency and the relative size of the components of the biphasic response of the vas deferens to electrical stimulation at different temperatures.

1. Electrical stimulation of the guinea-pig or rat vas deferens (pre- or post-ganglionically) at frequencies from 2-5 to 40 Hz with trains of stimuli of 30 sec duration induced a biphasic response. A rapid contraction (component A) was followed after a brief relaxation by a slower contraction (component B); the two phases were seen most clearly with stimulation frequencies of less than 10 Hz. 2. The responses to post-ganglionic stimulation were always larger than those to preganglionic stimulation. In general, at low frequencies component A exceeded component B whilst at high frequencies component B was the larger. Separation of the two components on the basis of their frequency response characteristics was better for rat than for guinea-pig vasa. 3. Log. frequency-response curves to transmural (post-ganglionic) electrical stimulation and log dose-response curves to noradrenaline were recorded for guinea-pig and rat vasa deferentia at 32 degrees, 22 degrees and 12 degrees C. For the guinea-pig reduction of bath temperature to 12 degrees C increased the amplitude of component A at 2-5 and 5 Hz; component B could not confidently be distinguished at this temperature. At 22 degrees C there was potentiation of B at lower frequencies and depression of B at higher frequencies. There was no response to noradrenaline at 12 degrees C. At 22 degrees C the response to noradrenaline was increased except to doses at or near the maximum to which the response was reduced. 4. For the rat was deferens component A was little changed by reduction of temperature. Component B at 12 degrees C was greatly depressed at higher frequencies. The response to noradreanaline was increased to lower doses and decreased to higher doses as the temperature was lowered. 5. The B component of the response of guinea-pig vasa at 22 degrees C and rat vasa at 32 degrees C was more sensitive than the A component to inhibition by thymoxamine. 6. Further analysis of the mechanisms underlying the A and B components of the biphasic response may be facilitated by relative isolation of each component by the appropriate selection of parameters of electrical stimulation and of temperature for the species being investigated. The contractions of the B component are similar to, if not identical with, those produced by exogenously applied noradrenaline.     

Transmural field stimulation-induced relaxation in the rat common hepatic artery.

Hepatic arteries are reportedly innervated by vasoconstrictor and vasodilator nerves. Experiments were carried out to investigate the possible involvement of calcitonin gene related peptide (CGRP) and nitric oxide as neurotransmitters during the relaxation of the rat common hepatic artery produced by transmural electrical field stimulation (ES). Common hepatic arteries were excised under ether-anesthesia from 6 weeks-old female rats, and isometric tensions recorded from endothelium-damaged ring preparations. In the presence of atropine and guanethidine, ES relaxed arteries which had been previously contracted with vasopressin. The relaxation response to ES was attenuated by either tetrodotoxin or capsaicin-pretreatment. CGRP induced a concentration-dependent relaxation, which was inhibited by the CGRP antagonist CGRP(8-37). The ES-induced relaxation was attenuated either slightly by the nitric oxide synthesis inhibitor L-nitroarginine (L-NNA) or markedly by CGRP(8-37). The relaxation response was nearly abolished in the presence of both CGRP(8-37) and L-NNA. These results may indicate that the nerve stimulation-induced vasodilatation of the rat common hepatic artery is mediated mainly by CGRP and partly by nitric oxide.