Pharmacological analysis of functional neurovascular transmission in canine splenic arteries: role of neuropeptide Y

1 The effects of neuropeptide Y (NPY) upon the isolated vasculature are reviewed. 2 The vasconstrictor responses to periarterial nerve stimulation (PNS) and neurotransmission by noradrenaline (NA) and ATP are discussed and illustrated using canine isolated perfused splenic artery. 3 Modulation of the vascular responses to PNS by NPY via pre- and post-junctional NPY Y2 and Y1 receptors is discussed. 4 Evidence is presented for different alpha1-adrenoceptor subtypes mediating vasoconstriction to exogenous and endogenously released NA and their different locations in the neurovascular junction and extrajunctional regions. 5 Activation of NPY Y1-receptors potentiates sympathetic nerve activated alpha1-adrenoceptor vasoconstriction. The proposal that the postjunctional alpha1B adrenoceptor may be linked to the NPY Y1-receptor and is responsible for co-operation between sympathetic and NPYergic interactions in the vasculature is discussed.     

EFFECTS OF ENDOTHELIUM-DERIVED RELAXING FACTOR ON THE SMOOTH MUSCLE OF THE RAT TAIL ARTERY

Simultaneous measurements of smooth muscle membrane potential and tension were made from isolated pieces of rat tail artery. A single electrical stimulus to the perivascular nerves produced a transient contraction of the smooth muscle. The amplitude of the contraction was increased after removal of the endothelium. The excitatory junction potentials and action potentials in the smooth muscle had the same amplitudes before and after removal of the endothelium. Tension obtained by direct stimulation of the arterial muscle in guanethidine-treated arteries was also increased by removal of the endothelium. When the artery was constricted by noradrenaline or 5-hydroxytryptamine, electrical stimulation caused a relaxation that was reduced by removing the endothelium. It was concluded that the electrical stimulus released the endothelium-derived relaxing factor (EDRF) which reduced the amount of contractile force that could be produced by an action potential in the smooth muscle.     

Pharmacological studies of piperine; I. Effects of piperine on transmural nerve stimulation.

The effects of pipeline on the responses of the fruit-bat and guinea pig gastrointestinal tract smooth muscle to transmural nerve stimulation (TNS) were studied. Piperine (all concentrations) reduced or abolished the responses of the bat oesophagus, duodenum, rectum and the guinea pig stomach. Low concentrations enhanced the responses of the guinea pig oesophagus and ileum whilst higher concentrations produced inhibition. Bat ileum showed four types of responses to TNS: contraction plus relaxation; contraction only; relaxation followed by contraction and relaxation. The contractions were abolished by tetrodotoxin whilst the relaxations were resistant. Piperine blocked the contractions but the relaxations were not readily blocked. It is concluded that piperine is a non-specific spasmolytic.     

The presynaptic activity of huwentoxin-I, a neurotoxin from the venom of the chinese bird spider Selenocosmia huwena.

Three different types of isolated nerve-synapse preparations, guinea pig ileum, rat vas deferens and toad heart, were used to investigate the physiological activity of Huwentoxin-I, a neurotoxin from the venom of the spider Selenocosmia huwena. The twitch response of isolated guinea pig ileum induced by electrical stimulus can be inhibited by HWTX-I. After blockage, contraction of the ileum can be induced by exogenously applied acetylcholine. HWTX-I caused the inhibition of the twitch response to electrical nerve stimulation in the rat vas deferens. After the twitch was completely inhibited, noradrenaline triggered rhythmic contraction of the vas deferens. The inhibitory effect on heart of toad induced by stimulating sympathetic-vagus nerve can be reversed by HWTX-I, although exogenously applied acetylcholine still acts as an effective inhibitor. All of these results support the conclusion that HWTX-I has the presynaptic activity that effects the release of neurotransmitter from the nerve endings of both the cholinergic synapse and the adrenergic synapse.     

Neuronal influence on the mechanical activity of the ciliary muscle.

1 Neuronal effects and the pharmacological properties of the bovine ciliary muscle were investigated in vitro. The bovine ciliary muscle exhibited no spontaneous activity. 2 Electrical stimulation of an isolated short ciliary nerve produced distinct contractions. The minimal stimulus duration required to evoke a contraction was 0.2 ms and amplitude of the contraction was maximal at 2 ms. Twitch or incomplete tetanus reached a complete tetanus with 4 Hz stimulation. 3 Raising the external potassium concentration from 5.9 to 158.8 mM produced a contracture which consisted of an initial phasic and then tonic components. 4 The contractions generated by either electrical stimulation (0.2-100 ms) or high K were potentiated by physostigmine and completely inhibited by atropine. Neither adrenoceptor agonists nor blockers influenced these contractions. 5 Application of tetraethylammonium (TEA), potentiated the electrically-induced ciliary muscle contraction, and the effect of TEA was not completely inhibited by high concentrations of either atropine or tetrodotoxin. Thus, TEA presumably acts both pre-junctionally and post-junctionally to increase the contractile development of ciliary muscle. 6 The ciliary contractile response is primarily mediated by acetylcholine released from nerves, and this response is accompanied by a negligible contribution from the sympathetic nerves. Depolarization induced by electrical currents or by high K was ineffective in evoking contraction of the ciliary muscle. 7 The results suggest that excitation of the ciliary muscle is probably mediated via junction potentials or by a direct transmitter action without any very great change in the potential. Action potentials are probably generated in the presence of TEA.     

Linopirdine (DuP 996) selectively enhances acetylcholine release induced by high potassium,but not electrical stimulation,in rat brain slices and guinea pig ileum

The enhancement of acetylcholine (ACh) release during electrical or potassium depolarization was compared in the presence of either linopirdine or 4-aminopyridine (4-AP) using two different functional assays. We measured ACh and choline (Ch) directly from striatal perfusates using high performance liquid chromatography and electrochemical detection or indirectly by measuring ACh-mediated twitch height changes in the electrically stimulated guinea pig ileum. Potassium-induced twitch responses were also measured in resting ileal segments. Linopirdine significantly enhanced ACh release in the presence of elevated potassium levels in rat striatal slices and guinea pig ileum, but had no effect during electrical stimulation in either model. In contrast, 4-AP enhanced ACh release in both electrically stimulated striatal slices and ileal strips. In agreement with other studies, atropine enhanced ACh release during either electrical or potassium stimulation paradigms in the striatal slice. The in vitro observations in this report suggest that linopirdine has limited ability to augment the cholinergic neurotransmission evoked by electrical stimulation. © 1993 Wiley-Liss, Inc.     

Antagonism of the ATP component of sympathetic co-transmission in the rat vas deferens by AMP

Field stimulation of isolated rat vas deferens preparations caused a twitch contraction followed by a slower phasic contraction. Exogenously applied ATP and noradrenaline caused rapid contractions and slowly developing contractions respectively. Pretreatment with phentolamine antagonised the phasic response to stimulation and the contraction to noradrenaline. Pretreatment with AMP antagonized the twitch response to stimulation and concentrations to ATP. The results support the view that ATP is released as a co-transmitter with noradrenaline in sympathetic nerves.     

Neuropeptide Y (NPY) reduces field stimulation-evoked release of noradrenaline and enhances force of contraction in the rat portal vein

The effect of neuropeptide Y (NPY) on fractional tritium-noradrenaline (3H-NA) release and contractile activity was studied in the isolated portal vein of SHR and WKY rats. NPY (5 X 10(-7) M) enhanced the force of the spontaneous contractile activity by about 40%. The fractional 3H-release elicited by transmural nerve stimulation (TNS), which mainly reflects 3H-NA, was reduced by about 40% after preincubation with 5 X 10(-7) M NPY in portal veins from both SHR and WKY rats. The inhibitory effect of NPY on TNS-evoked 3H-release was more slowly reversed by washout than the facilitatory action on spontaneous contractile force. The contractile response to field stimulation was not reduced by NPY, but rather tended to be increased. It is concluded that NPY exerts a dual action in the SHR and WKY portal vein, thus enhancing the smooth muscle contractions and inhibiting sympathetic neurotransmission. The inhibitory effect of NPY on TNS-evoked NA efflux, which is present in both SHR and WKY rats, is most likely due to a presynaptic site of action.     

In vitro action of uridine diphosphate glucose (UDPG) on phrenic diaphragm preparations

The action of uridine-5'-diphosphoglucose (UDPG) on the contractile response of the phrenic diaphragm preparation from guinea pig was investigated. UDPG activity was assayed on the preparation at rest or during the exercise; in this case indirect electrical stimulation of phrenic diaphragm preparation or direct stimulation of denervated or curarized muscle were employed. Krebs' solutions adequately modified with regard to glucose concentration were used. The effect of UDPG on the neuromuscular junction was also investigated by recording miniature end plate potentials. An effect of the drug on neuromuscular transmission and on glucose metabolism could be demonstrated.     

The role of prostaglandins in cholinergic neurotransmission in the guinea pig.

Prostaglandins have contrasting effects on neurotransmission at different cholinergic nerve endings. This is a report on the role of prostaglandins in a number of cholinergic preparations from the guinea pig. In the isolated ileum PGE1 (2 X 10(-10) to 5 X 10(-8) M) potentiated the response to electrical stimulation of the cholinergic nerves. PGE1 (10(-7 M) caused an increase in tone followed by a period of transient inhibition of twitch height. Responses to simulation of the ileum with drugs were not potentiated by PGE1. Responses of atropinized or plexus-free muscle to electrical stimulation were also not potentiated by PGE1. Acetylsalicylic acid (2.5 X 10(-4) M) diminished the twitch response and the output of acetylcholine from the ileum. Both effects were reversed by PGE1. Qualitatively similar observations were made on the trachea. It is concluded that prostaglandins facilitate acetylcholine release in the ileum and trachea. PGE1 diminished the effect of vagal stimulation on the heart rate. The response to stimulation of the phrenic nerve was not affected.     

Inhibition of cholinergic and non-adrenergic, non-cholinergic bronchoconstriction in the guinea pig mediated by neuropeptide Y and alpha 2-adrenoceptors and opiate receptors

The mechanisms underlying the regulatory influence of neuropeptide Y (NPY) and of alpha 2-adrenoceptor and opiate receptor activation on cholinergic and excitatory non-adrenergic, non-cholinergic (e-NANC) neurotransmission were studied in guinea pig hilus bronchi in vitro. NPY inhibited both the cholinergic and e-NANC bronchial contractions evoked by field stimulation. The NPY attenuation of the e-NANC contraction could not be antagonized by the alpha 2-antagonist, idazoxan, or naloxone. UK 14,304 a specific alpha 2-agonist, also reduced the two nervous components of bronchial contraction and this action was inhibited by idazoxan. NPY and UK 14,304 exerted a minor influence on the bronchial smooth muscle tone per se or on contractions evoked by acetylcholine or neurokinin A. This suggested that the inhibitory responses were caused by a prejunctional action reducing the release of transmitter substances from sensory and cholinergic nerve endings. Furthermore NPY (10(-7) M) seemed to be more potent to inhibit both contractile components than noradrenaline (10(-6) M) in the presence of propranolol (3 X 10(-6) M). Morphine was able to reduce the e-NANC response via a naloxone-sensitive mechanism. The capsaicin-evoked bronchoconstriction and the bronchodilator NANC effect evoked by field stimulation were, however, not influenced by UK 14,304. It is concluded that NPY, alpha 2-receptor and opiate receptor activation inhibit the release of sensory transmitters evoked by field stimulation but not by capsaicin.     

Non-cholinergic, non-adrenergic tonic component of the electrically-evoked contractions of guinea pig ileum

Contractile responses of the longitudinal muscle layer of guinea pig ileum to 40-sec electrical field stimulation (0.8 msec, 20 V) applied at frequencies of 5 Hz or 30 Hz consisted in a fast phasic contraction followed by a low-amplitude tonic component. Atropin (3 microM) and prazosine (1 microM) decreased significantly the amplitude of the phasic contraction and had less influence on the tonic component. SC 19220 (50 microM) additionally lowered both the fast and the tonic contractions, the inhibitory effect being more pronounced on the tonic component. It is suggested that prostaglandins released during electrical stimulation could be involved in the non-cholinergic, non-adrenergic tonic component of the contractile responses.     

Transmitter release and uptake evoked by the amphibian skin alkaloid,pumiliotoxin-B (PTX-B), in the electrically stimulated mouse vas deferens preparation (MVD)

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Histamine H3-receptors inhibit sympathetic neurotransmission in guinea pig myocardium.

The histamine H3 agonist, (R)-alpha-methylhistamine (alpha-MeHA, 10(-10) to 10(-5) M), caused a concentration-dependent inhibition of the sympathetic contractile response to electrical field stimulation of guinea pig isolated atria, but alpha-MeHA did not alter the basal tension or the contraction induced by exogenously applied norepinephrine. Blockade of H1 and H2 histamine receptors, and alpha- and beta-adrenoceptors failed to prevent the inhibitory effect of alpha-MeHA, whereas the specific H3 receptor antagonist, thioperamide, concentration dependently reversed the inhibitory effect of alpha-MeHA. At the concentration of 10(-7) M, which was effective for antagonizing the action of alpha-MeHA, thioperamide did not modify the sympathetic responses facilitated by the beta 2-adrenoceptor agonist, clenbuterol, or attenuated by the alpha 2-adrenoceptor agonist, clonidine. Our results suggest that H3 receptors exist on the cardiac sympathetic terminals, which may modulate adrenergic neurotransmission in guinea pig myocardium.     

Some pharmacological properties of piperazine

1. The action of piperazine on mammalian smooth, cardiac and skeletal muscles has been studied.2. Piperazine increased tone and produced a dose dependent contraction of isolated smooth muscle, which was antagonized by atropine.3. With cardiac muscle, piperazine depressed both the rate and force of contraction and was antagonized by atropine. At higher concentrations, a non-specific depression of cardiac muscle was found. The intravenous injection of piperazine produced a transient decrease in both heart rate and blood pressure and this was followed by an increase.4. In about half of the mammalian skeletal muscle preparations, piperazine potentiated the twitch of the muscle evoked by electrical stimulation.5. On the frog neuromuscular preparation, piperazine produced potentiation of the twitch but this was followed first by blockade of the effects of nerve stimulation and then by depression of the effects of direct muscle stimulation.     

Further studies on the effects of disodium cromoglycate on guinea pig ileum

Transmural electrical stimulation was carried out on innervated strips of the longitudinal muscle of guinea pig ileum. Disodium cromoglycate (DSCG) inhibited the electrically induced contractions. Five minutes later, prostaglandin E2 (2.5 ng/ml) was added to the bath and it reversed the action of DSCG. Furthermore, DSCG inhibits significantly the guinea pig ileum contractions induced by nicotine and also those induced by histamine and acetylcholine on ileum denervated by cooling. These results suggest that DSCG effects on guinea pig ileum contraction are mediated by membrane-stabilizing properties of this drug on smooth muscle fibres as well as on myenteric plexus.     

The effect of epibatidine on spontaneous and evoked neurotransmitter release in the mouse and guinea pig isolated vas deferens

BACKGROUND AND PURPOSE: Nicotinic agonists increase sympathetic field-stimulus-evoked contraction of the rodent vas deferens, presumably by increasing evoked neurotransmitter release. This presumption was tested in two species. EXPERIMENTAL APPROACH: The effect of the nicotinic acetylcholine receptor (nAChR) agonist epibatidine on neurotransmitter release in mouse and guinea pig isolated vas deferens was investigated using contraction studies and conventional intracellular recording techniques. KEY RESULTS: In 12 of 14 mouse vasa deferentia, slow bath application of epibatidine (100 nM) had no significant effect on excitatory junction potential (EJP) amplitude and spontaneous EJP (SEJP) frequency. However, rapid application of epibatidine to the mouse vas deferens caused an increase in SEJP frequency (by 530%), with no effect on EJP amplitude. Despite the absence of an effect on EJPs, electrically-evoked contractions of the mouse vas deferens were significantly increased in the presence of epibatidine (by 50%). A transient contraction was reliably induced by a higher epibatidine concentration (1 microM). This contraction was significantly reduced in the presence of prazosin, tetrodotoxin, or alpha,beta-methyleneATP. Epibatidine did not induce a contraction in the presence of a combination of prazosin, alpha,beta-methyleneATP and cyclopentolate. In guinea pig vasa deferentia, bath-applied epibatidine potentiated EJP amplitude in a biphasic pattern, lasting for at least 30 minutes. CONCLUSION AND IMPLICATIONS: The nAChR-mediated augmentation of neurogenic contraction is indeed prejunctional, but in the mouse arises from an increase in spontaneous neurotransmitter release that primes smooth muscle for subsequent contraction, while in the guinea pig there is a direct augmentation of evoked neurotransmitter (ATP) release.     

Pharmacological characterisation of the smooth muscle antispasmodic agent tiropramide

(+/-) Tiropramide hydrochloride, its D and L optical isomers and some of its metabolites were characterized in a number of in vitro pharmacological tests. Tiropramide showed broad spectrum antispasmodic activities on the isolated stomach of guinea pig electrically stimulated; on the longitudinal muscles of the ileum of guinea pig stimulated by electrical impulses, BaCl2, acetylcholine, histamine, serotonin, substance P and cholecystokinin octapeptide (CCK-8); on the spontaneous contractions and on the electrical inhibition of the jejunum of rabbit; on the spontaneous contractions and on the contractions provoked by BaCl2 and acetylcholine of the ascending colon of the rat; on the contractions provoked by BaCl2, acetylcholine, histamine and cerulein of the circular muscles of the gall bladder of the guinea pig; on the spontaneous contractions of the pyel-ureter preparation of the guinea pig; on the contractions of the uterus of the rat provoked by oxitocin, serotonin, acetylcholine, PGF2; on the spontaneous contraction of the portal vein of the rat; on the constriction of the tail artery of the rat provoked by electrical stimulation, epinephrine and ergotamine; on the contractions of the aortic strip of the rabbit stimulated by norepinephrine; on the contractions of the strip of bovine coronary artery depolarized by HCl. In general tiropramide had antispasmodic effect at 5-60 mumol/l concentration. It was more potent than papaverine on contractions provoked by electrical or chemical stimuli, and was less potent or ineffective on spontaneous and "physiological" contractions of the different smooth muscle preparations. Tiropramide had small effects on vascular smooth muscles and showed very small calcium channel blocking activity.     

Neuropeptide Y1- and Y2-receptor-mediated cardiovascular effects in the anesthetized guinea pig, rat, and rabbit

Neuropeptide Y (NPY) causes vasoconstriction through Y1-receptors and inhibits vagal bradycardia through presynaptic Y2-receptors. These effects of NPY were investigated in anesthetized guinea pigs, rats, and rabbits to find the most suitable species for evaluation of Y1- and Y2-active agents in vivo. The increase in blood pressure (through Y1) of lower doses of NPY was similar in the three species (ED50, 0.9 +/- 0.13, 0.8 +/- 0.39, and 0.6 +/- 0.09 nmol/kg, respectively), but higher doses had depressor effects in four of six rats. Vagal bradycardia, induced by electrical stimulation of the right cervical vagus nerve, was inhibited by NPY in the guinea pig and in the rat (ED35, 3.5 +/- 0.46 and 11.2 +/- 1.79 nmol/kg, respectively; p < 0.05) but not in the rabbit. In the guinea pig, the Y2-receptor-preferring fragment NPY(3-36) and the selective Y1-receptor antagonist H 409/22 were used to confirm that the increase in blood pressure was mediated solely through the Y1-receptor and the vagal inhibition solely through the Y2-receptor. Aside from the cardiovascular effects, NPY caused a decrease in the body temperature and inhibited vagal bronchoconstriction in this species. Considering that NPY may cause depressor effects in the rat and has no effect on the vagal bradycardia in the rabbit, the guinea pig is preferable to both these species for assessment of Y1- and Y2-receptor-active agents in vivo.     

Pharmacological profile of the novel alpha-adrenoceptor antagonist KT-611 (naftopidil).

The pharmacological profile of a new alpha-adrenoceptor antagonist, KT-611 (naftopidil), was studied in vitro. In the dog mesenteric and carotid arteries and in the rabbit, guinea pig and rat thoracic aortae, KT-611 competitively inhibited alpha 1-adrenoceptor-mediated contractions induced by noradrenaline with pA2 values ranging from 6.73 to 8.15. KT-611 also inhibited the postjunctional alpha 2-adrenoceptor-mediated contractions in the dog saphenous vein (pA2 = 6.77) or dog basilar artery. However, the responses mediated through prejunctional alpha 2-adrenoceptors (rat vas deferens), beta-adrenoceptors (rat atria), muscarinic receptors (guinea pig ileum) and 5-HT2 receptors (dog mesenteric artery) were little affected by KT-611. KT-611 also inhibited the sympathetic adrenergic contraction evoked by electrical transmural stimulation in the dog mesenteric artery, and the inhibition was not relieved upon repetitive washing for 1 hour with the drug-free solution. 3H-prazosin and 3H-clonidine binding to the rat cortex membranes was inhibited by KT-611 with pKi values of 7.69 and 5.75, respectively. These results suggest that KT-611 is an alpha 1-adrenoceptor antagonist with a weak antagonistic activity to postjunctional alpha 2-adrenoceptors.