Single Pulse Field Stimulation Research Articles

Ketanserin potentiates the prejunctional inhibitory effect of 5-hydroxytryptamine on rat vas deferens.

5-Hydroxytryptamine (5-HT) slightly inhibited the twitch contractions of rat vas deferens caused by single pulse field stimulation at 0.1 Hz. The inhibitory effect of 5-HT was much less in the epididymal portion than in the prostatic portion of the vas deferens. Ketanserin potentiated the prejunctional inhibitory effect of 5-HT and attenuated its stimulatory effect. This potentiation was observable only in the epididymal portion, of the vas deferens. Cyproheptadine and mianserin, but not methysergide, had essentially similar potentiating effects to those of ketanserin. These results suggest that the 5-HT receptor that mediates prejunctional inhibition is not of the 5-HT2 type, and that ketanserin acts by suppressing the 5-HT-induced stimulatory effect, which is possibly mediated by a postjunctional 5-HT2 receptor, thus unmasking the inhibitory effect of 5-HT.

Cromakalim blocks the purinergic response evoked in rat vas deferens by single-pulse electrical stimulation

The present study was carried out to look at the influence of the K + channel opener cromakalim, compared with suramin and prazosin, on the contractile response evoked by single-pulse field stimulation and exogenous agonists in epididymal and prostatic portions of rat vas deferens. In the epididymal portion suramin abolished the first phase of the response to single shock, prazosin deeply affected the second phase and a combination of both antagonists almost completely abolished both phases. Cromakalim was able to inhibit in a concentration-dependent manner the first purinergic phase (pD 2 = 5.90 ± 0.11), leaving practically unaffected the second, adrenergic phase. This inhibitory effect of cromakalim on the electrically evoked response was counteracted by glibenclamide. Cromakalim and prazosin, but not suramin, affected the response to exogenous noradrenaline. Suramin but not cromakalim was able to antagonize responses to α,β-methylene-ATP. In the prostatic portion because of a less clear discrimination between adrenergic and purinergic phases of the electrically evoked response, the picture was less clear although the trend was identical. Cromakalim was not able to antagonize the response to ATP. It is concluded that in rat vas deferens cromakalim inhibits purinergic transmission by acting prejunctionally.

Suramin: a selective inhibitor of purinergic neurotransmission in the rat isolated vas deferens

The effects of the putative P 2 purinoceptor antagonist suramin on contractile responses of the rat isolated vas deferens to electrical field stimulation and exogenously applied drugs (α,β-methylene ATP and noradrenaline) were investigated. Suramin was devoid of agonist activity in the rat vas deferens. The response of the vas deferens to single pulse field stimulation was characteristically biphasic with a large first component peaking between 260–300 ms after the stimulus followed by a second smaller component peaking at about 650 ms post-stimulus. Suramin (100 nM-1 mM) selectively impaired the first, purinergic phase of the response to single pulse field stimulation but was without effect on the second, noradrenergic component. The response of the vas deferens to trains of electrical field stimuli (10 Hz for 10 s) was also biphasic. Suramin (1 μM-1 mM) reduced the first (< 1 s) phase of the response by 30%, the second (< 5s) plateau phase by 50% and inhibited the intermediate (2–4 s) phase by 80%. Dose-contact periods of 20 or 30 min respectively were sufficient to achieve equilibration of the inhibitory effects of suramin against the responses to single pulse field stimulation or trains of pulses. Following 30 min incubation with 1 mM suramin, the remaining first and second phase components of the response to trains of pulses were impaired and subsequently abolished by the α 1-adrenoceptor antagonist WB4101 establishing their noradrenergic origin. Suramin (300 μM) abolished responses of the vas deferens to α,β-methylene ATP but was without effect on those to noradrenaline. Suramin (30 μM) induced a rightward shift in the concentration-response relationship to α,β-methylene ATP that was associated with a significant 40% increase in the maximum response, but did not modify responses to noradrenaline. The inhibitory effects of suramin (3–300 μM) on responses of the vas deferens to approximate EC 50 concentrations of α,β-methylene ATP were reversible on repeated washout for 40–60 min. These results reveal suramin to be a useful pharmacological tool for the study of purinergic neurotransmission in rodent vasa deferentia.

Modulation of cholinergic neurotransmission by the peptide VIP, VIP antiserum and VIP antagonists in dog and cat trachea.

1. Comparative studies on the effects of vasoactive intestinal polypeptide (VIP), commercially available VIP antiserum or VIP antagonists [Ac-Tyr1, D-Phe2]-GRF(1-29)-NH2 and [4-Cl-D-Phe6, Leu17]-VIP on excitatory neuroeffector transmission in the dog and cat trachea were performed with microelectrode, double sucrose-gap, and tension recording methods. 2. VIP (10(-11)-10(-9) M) had no effect on the resting membrane potential or on the input resistance of the smooth muscle cells of dog and cat trachea. However, with increased concentrations (greater than 10(-8) M) VIP hyperpolarized the membrane and decreased the input resistance of the membrane in both tissues. 3. VIP (10(-10)-10(-7) M) dose-dependently reduced the amplitude of the contractions evoked through the nervous structure excited by field stimulation in the combined presence of indomethacin (10(-5) M) and guanethidine (10(-6) M) in the dog, and in the presence of guanethidine (10(-6) M) in cat trachea. In parallel with actions on twitch contractions, VIP (10(-11)-10(-7) M) reduced the amplitude of the excitatory junction potentials (EJPs) evoked through the nervous structure excited by single pulse field stimulation in both tissues. 4. VIP (10(-9) M) had no effect on the post-junctional response of smooth muscle cells to exogenous acetylcholine (ACh) (10(-9)-10(-5) M). 5. During repetitive field stimulation at the stimulus frequency of 0.033-0.1 Hz, the amplitude of the EJPs was gradually reduced, and VIP (10(-9) M) enhanced this depression phenomenon in the dog and cat trachea. 6. EJPs also showed summation when repetitive field stimulation was applied at high frequency (20 Hz) in the dog trachea. The slope of the relationship between the relative amplitude of the EJP and number of stimuli at 20 Hz was 2.2 +/- 0.4 mV/stimulation (n = 4) in the dog trachea. However, in the cat trachea, summation of EJPs was not prominent, giving a mean slope of 0.6 +/- 0.2 mV/stimulation (n = 6) measured by the microelectrode method. VIP (10(-9) M) shifted downward the relationship between the relative amplitude of the EJP and the number of stimuli at 20 Hz in both tissues. 7. Overnight incubation with VIP antiserum (10(-6) g/ml) had little effect on the depression of the EJP in the dog and cat trachea, or the summation of the EJP observed in the dog trachea.(ABSTRACT TRUNCATED AT 400 WORDS)

Pre- and post-junctional actions of procaterol, a beta 2-adrenoceptor stimulant, on dog tracheal tissue.

1. The effects of procaterol, a beta 2-adrenoceptor agonist, on excitatory neuro-effector transmission in the dog trachea were investigated and the findings were compared to those seen with isoprenaline, with microelectrode, double sucrose gap and tension recording methods. 2. Procaterol (10(-10)-10(-9) M) and isoprenaline (10(-9) M) had no effect on the resting membrane potential or on the input resistance of the smooth muscle cells of dog trachea. However with increased concentrations (greater than 10(-8) M), these agents hyperpolarized the membrane and decreased the input resistance of the membrane. 3. Procaterol (10(-10)-10(-7) M) and isoprenaline (10(-9)-10(-7) M) dose-dependently reduced the amplitude of the twitch contractions evoked by field stimulation in the combined presence of indomethacin (10(-5) M) and guanethidine (10(-6) M). In parallel with actions on twitch contractions, procaterol (10(-10)-10(-7) M) and isoprenaline (10(-9)-10(-7) M) reduced the amplitude of the excitatory junction potentials (e.j.ps), evoked by single pulse field stimulation in the dog trachea. 4. Procaterol (10(-8) M) had no effect on the post-junctional response of smooth muscle cells to exogenous acetylcholine (ACh) (10(-7)-10(-6) M). 5. Pretreatment with ICI-118551, a beta 2-adrenoceptor blocking agent, reduced the inhibitory action of procaterol on the amplitude of twitch contractions evoked by field stimulations in the dog trachea. 6. These results indicate that procaterol in low concentrations has a prejunctional action inhibiting the excitatory neuro-effector transmission in addition to a postsynaptic action, presumably by suppressing transmitter release from the vagus nerve terminals through beta 2-adrenoceptors in the dog tracheal tissue. The pre- and post-junctional actions of procaterol explain its potent bronchodilator effects in clinical use.

Age-related change in serotonin-mediated prejunctional inhibition of rat vas deferens

The effect of age on the serotonin (5-HT)-induced prejunctional inhibition of twitch contractions induced in rat vas deferens by single-pulse field stimulation at 0.1 Hz was studied. The inhibitory effect of 5-HT gradually decreased with increasing age of the rats (4–15 weeks old) and was no longer detectable in preparations from rats over 15 weeks old. Moreover, on a further increase in age to 45 weeks, 5-HT conversely enhanced the twitch response of the vas deferens. The 5-HT 2 antagonists ketanserin and mianserin potentiated the 5-HT-induced inhibition of contractions of the vas deferens of young rats (8–15 weeks old), and attenuated the amplifying effect of 5-HT on the responses of preparations from old rats (95 weeks old). Thus unmasking of the inhibition depended on the age of rats. This change occurred earlier in life in the prostatic portion of the vas deferens than in the epididymal portion. A functional decrease in 5-HT-mediated prejunctional inhibition and the resultant increase in amplification of the twitch response by 5-HT are probably responsible for the age-related decrease in prejunctional inhibition.

REDUCED α1‐ADRENORECEPTOR MEDIATED RESPONSIVENESS IN VAS DEFERENS FROM SPONTANEOUSLY HYPERTENSIVE RATS

alpha-adrenoreceptor-mediated responses were investigated in isolated vasa deferentia from spontaneously hypertensive rats (SHR), Wistar Kyoto rats (WKY) and normotensive rats (NWR). There was no significant difference between NWR, WKY and SHR in the inhibition of the isometric contraction to single pulse field stimulation by the alpha 2-selective agonist xylazine in prostatic portions, nor by xylazine and the alpha 1-selective agonist amidephrine in epididymal portions in the presence of nifedipine to prevent postjunctional actions of alpha 1-selective agonists. There was no significant difference between NWR, WKY and SHR in the potency of amidephrine in causing a postjunctionally mediated potentiation of the isometric contraction to single pulse field stimulation in prostatic portions but the maximum potentiation was significantly reduced in SHR. However contraction by the calcium entry facilitator, Bay K 8644, was not significantly different between NWR, WKY and SHR. The maximum direct contraction to amidephrine, but not to Bay K 8644, was also significantly reduced in SHR. It is concluded that the altered alpha 1-adrenoreceptor-mediated responsiveness in SHR is due not to genetic strain, but is presumably linked to development of hypertension.

An investigation of α‐adrenoceptor responsiveness in the vas deferens of spontaneously hypertensive rats

alpha-Adrenoceptor-mediated responses were investigated in isolated vasa deferentia from spontaneously hypertensive rats (SHR) and normotensive Wistar rats (NWR). There was no significant difference between NWR and SHR in the inhibition of the isometric contraction to single pulse field stimulation by alpha 2-selective agonists in prostatic portions, nor by alpha 2-selective agonists and the alpha 1-selective agonist, amidephrine in epididymal portions in the presence of nifedipine to prevent postjunctional actions of alpha-1-selective agonists. There was no significant difference between NWR and SHR in the potency of amidephrine in causing a postjunctionally mediated potentiation of the isometric contraction to single pulse field stimulation in prostatic portions but the maximum potentiation was significantly reduced in SHR. However, the maximum potentiation of the isometric contraction by the calcium entry facilitator, Bay K 8644, was not significantly different between NWR and SHR. The maximum direct contraction to amidephrine, but not to Bay K 8644, was also significantly reduced in SHR. The irreversible alpha 1-adrenoceptor antagonist, phenoxybenzamine was more potent in SHR than NWR in reducing the maximum potentiation by amidephrine of the stimulation-evoked isometric contraction and in reducing the maximum direct contraction to amidephrine. It is concluded that there is a reduced postjunctional alpha 1-mediated responsiveness in the vas deferens of SHR due probably to a reduction in receptor number.

Further examination of the effects of ageing on the adrenoceptor responsiveness of the rat vas deferens

Age-related changes in presynaptic α 2-adrenoceptors were investigated in prostatic and epididymal portions of vasa deferentia from young adult (2–3 months) and old (24–29 months) Sprague-Dawley rats, using the α 2-selective agonists xylazine and clonidine. In prostatic portions the inhibitory effects of clonidine against the isometric contractions to single pulse field stimulation were complicated by a postsynaptic action in old animals, but in epididymal portions in the presence of nifedipine both xylazine and clonidine were 3 times less potent in old rats. However, there were no significant differences between young and old in the potency of xylazine at inhibiting the overflow of tritium or the isometric contraction evoked by 5 Hz stimulation for 3 min in tissues pre-incubated with [ 3]Hnoradrenaline. It is suggested that there is reduced responsivenes of presynaptic α 2-adrenoceptors in the vas deferens of old rats, but that this can only be demonstrated using a sensitive measure of the presynaptic potency of agonists: the isometric contraction to a single stimulus.

The actions of cirazoline on the rat vas deferens.

The pre- and postsynaptic effects of the alpha 1-agonist cirazoline were assessed in epididymal and prostatic portions of the rat isolated vas deferens. Cirazoline produced a postsynaptic alpha 1-adrenoceptor mediated potentiation of the isometric contraction to single pulse field stimulation in both prostatic and epididymal portions. In epididymal portions, nifedipine (10 microM) greatly attenuated the postsynaptic alpha 1-receptor mediated potentiation of nerve mediated contractions, uncovering a presynaptic inhibitory action of cirazoline . No evidence was found for alpha 2-antagonism by cirazoline . It is concluded that the previously reported antagonism of the presynaptic inhibitory effects of clonidine was due to postsynaptic potentiation of nerve-mediated responses by cirazoline .

Post-natal development of functional neurotransmission in rat vas deferens.

Responses of the rat vas deferens to drugs and to field stimulation were examined in sexually immature rats. The vasa from immature rats often exhibited spontaneous contractions and displayed greater sensitivity to the contractile effects of alpha-adrenoceptor agonists. The responses of the vasa from immature rats to single pulse field stimulation lacked the adrenergic component of the response although the non-adrenergic component was present. The responses were antagonized by alpha 2-adrenoceptor agonists. In the presence of cocaine, an adrenergic component of the response did appear. During trains of pulses the pre- and postjunctional effects of adrenergic transmission which are found in adult rats were absent in vasa from immature rats. Electron microscopic studies showed no qualitative differences in adrenergic innervation in vasa from immature and adult rats. It is concluded that a state of 'pre-innervation supersensitivity' associated with a lack of functional adrenergic transmission exists in the vas deferens of immature rats. The supersensitivity disappears and functional transmission develops during the period in which testosterone secretion increases in the rat. The reason for the lack of functional transmission at a time when the innervation appears to be morphologically mature is not clear but may be due to the noradrenaline release mechanism not being fully operative.

An examination of age-related changes in pre- and postsynaptic α-adrenoceptors in the rat isolated vas deferens

Age-related changes in pre- and postsynaptic α-adrenoceptors were investigated in prostatic and epididymal portions of vasa deferentia from young adult (2–3 month) and 24 month old Sprague-Dawley rats, using the selective α 1-agonist amidephrine and the selective α 2-agonist xylazine. Effects of agonists were assessed against the isometric contraction to single pulse field stimulation. There was no change in the pre- or postsynaptic potency of amidephrine, but the presynaptic inhibitory potency of xylazine was significantly reduced in old rats. Therefore, in old rats there is a reduced sensitivity of presynaptic α 2- but no change in pre- or postsynaptic α 1-adrenoceptors and this may result in increased peripheral sympathetic neurotransmission.

A COMPARISON OF THE EFFECTS OF NIFEDIPINE AND VERAPAMIL ON RAT VAS DEFERENS

Nifedipine preferentially blocks contractions of the prostatic end of the rat vas deferens to single pulse field stimulation, leaving the epididymal end largely unaffected. This action is not due entirely to antagonism of calcium influx. Verapamil unexpectedly potentiated the responses of the prostatic portion, and antagonized those of the epididymal end. The use of nifedipine may, therefore, allow investigations of adrenergic mechanisms on this tissue to be studied without the complications of non-adrenergic transmission.

The distribution of adrenoceptors and other drug receptors between the two ends of the rat vas deferens as revealed by selective agonists and antagonists.

1. The effects of adrenoceptor agonists and other agonists on the contractile responses of the prostatic and epididymal portions of the rat isolated vas deferens to single pulse field stimulation were investigated. 2. alpha-Adrenoceptor agonists produced prejunctional alpha 2-mediated inhibition and post-junctional alpha 1-mediated potentiation of the nerve-induced responses. Guanabenz and xylazine produced mainly inhibitory effects, xylazine being 10 times less potent. Clonidine and oxymetazoline produced inhibition with similar potency to guanabenz but at higher concentrations excitatory effects were present, particularly in the epididymal portion. Phenylephrine produced only potentiation of the nerve-induced response in both portions. Potentiation of nerve-induced responses was a more sensitive and quantitative index of excitation than was direct contraction of the tissue. 3. Isoprenaline and salbutamol both gave beta 2-mediated inhibition of the nerve-induced responses in both portions of tissue. At least part of the effect was post-junctional since phenylephrine contractions were inhibited. Isoprenaline also produced a post-junctional alpha 1-mediated excitation. 4. Noradrenaline produced effects qualitatively similar to those of the other alpha-adrenoceptor agonists, inhibition and excitation predominating in the prostatic and epididymal ends respectively. 5. Morphine produced inhibition in the mouse but not in the rat vas deferens. In rat vas, however, enkephalin analogues produced pre-junctional inhibition of responses in both portions which could be partly reversed by naloxone; restoration of the adrenergic component was more complete. Rat anococcygeus showed no equivalent effect. 6. Adenosine 5'-triphosphate (ATP) inhibited nerve-induced responses in each portion with a greater effect on the prostatic portion.

AN INHIBITORY ROLE FOR NORADRENALINE IN THE MOUSE VAS DEFERENS

1 Noradrenaline (0.1-3.0 muM) inhibited the twitch responses to single pulse field stimulation of the isolated vas deferens of the mouse. The higher concentrations of noradrenaline (ca. 0.3-3.0 muM) were required to make the tissue contract.2 Phentolamine (10 muM) abolished the contractor response to higher concentrations of noradrenaline and antagonized the inhibitory effect of lower concentrations on the twitch response.3 Propranolol (10 muM) potentiated both the contractor and the inhibitory effect of noradrenaline on the twitch response.4 Isoprenaline (0.1-3.0 muM) and salbutamol (1.0-3.0 muM) both inhibited the twitch response. Their effects were antagonized by propranolol (10 muM), but not by practolol (10 muM).5 The effects of uptake(1) and uptake(2) blocking agents were determined. Cocaine (10 muM) reduced the size of the twitch response in 2 out of 4 experiments. Imipramine (0.18 muM) also reduced the size of the twitch, as did oestradiol (3.7 muM) and a combination of cocaine and oestradiol.6 Contractor responses to exogenous noradrenaline showed tachyphylaxis, but when this was not very marked, the response could be shown to be potentiated by uptake blocking agents.7 The inhibitory effect of noradrenaline on the twitch response was greatly potentiated by cocaine (10 muM) and much less so by oestradiol (3.7 muM).8 It is concluded that the transmitter responsible for the twitch response is either an unknown substance released from the sympathetic neurone, or noradrenaline acting upon a receptor with none of the characteristics of known alpha- or beta-adrenoceptors. In either case, noradrenaline can inhibit the output, probably by stimulation of presynaptic alpha-adrenoceptors.