Interaction of prostaglandins and adenosine 5′-triphosphate in the noncholinergic neurotransmission in rabbit detrusor

Part of the excitatory transmission in rabbit detrusor is noncholinergic and nonadrenergic, and prostaglandins (PGs) and adenosine 5′-triphosphate (ATP) have been implicated in this transmission. The present experiments investigate the possibility of an interaction between PGs and ATP in rabbit detrusor. Indomethacin (2.8 μM) depressed the contraction produced by ATP although it did not antagonize the contraction produced by ATP although it did not antagonize the contraction produced by carbachol. Treatment of detrusor strips with 1.5 mM ATP depressed the frequency response curve in field stimulated tissues. This depression was additive with that produced by atropine. In the present experiments indomethacin did not significantly augment the effect of desensitization with ATP. It is suggested that the atropine-resistant neurotransmission in rabbit detrusor may involve both ATP and PGs acting in cooperation.     

Interaction of prostaglandins and adenosine 5'-triphosphate in the noncholinergic neurotransmission in rabbit detrusor.

Part of the excitatory transmission in rabbit detrusor is noncholinergic and nonadrenergic, and prostaglandins (PGs) and adenosine 5'-triphosphate (ATP) have been implicated in this transmission. The present experiments investigate the possibility of an interaction between PGs and ATP in rabbit detrusor. Indomethacin (2.8 muM) depressed the contraction produced by ATP although it did not antagonize the contraction produced by carbachol. Treatment of detrusor strips with 1.5 mM ATP depressed the frequency response curve in field stimulated tissues. This depression was additive with that produced by atropine. In the present experiments indomethacin did not significantly augment the effect of desensitization with ATP. It is suggested that the atropine-resistant neurotransmission in rabbit detrusor may involve both ATP and PGs acting in cooperation.     

Multiple responses to electrical field stimulation in circular muscle of canine gastric corpus

Innervation of circular muscle of the canine stomach studied in vitro was investigated by subjecting muscle strips to electrical field stimulation. Strips were cut from the lesser curvature of the gastric corpus and stimulated with 10-s trains of 0.5-ms pulses at 0.5-20 Hz, 40 V. Most responses were classified into one of three types. In general, field stimulation tended to elicit sequences of varying magnitudes of transient on-contraction, on-relaxation, off-relaxation, off-contraction. Responses were abolished by tetrodotoxin. On-contraction was almost abolished by atropine plus desensitization by 5-hydroxytryptamine (5-HT) or substance P. On-relaxation and off-relaxation were not affected by adrenergic blockade, methysergide, apamin, or 4-aminopyridine. ATP usually caused contraction and slightly diminished relaxation to field stimulation. Vasoactive intestinal polypeptide (VIP) had little effect on tone and response to field stimulation. Relaxation disappeared after scorpion venom treatment. This probably resulted from depletion of the transmitter which mediates relaxation. Off-contraction was reduced by atropine, desensitization by 5-HT or substance P, cromoglycate, indomethacin or ATP, but was not affected by adrenergic blockade, hexamethonium, methysergide, mepyramine, or VIP. The findings suggest that innervation of gastric corpus circular muscle included excitatory cholinergic and both excitatory and inhibitory noncholinergic, nonadrenergic innervation. However, the responses of circular muscle to field stimulation in vitro were drastically different from those obtained previously in vivo, suggesting damage or altered inputs to circular muscle when strips of circular muscle are studied.     

Trigeminal nerve: the possible origin of substance p-nergic response in isolated rabbit iris sphincter muscle

We determined the effects of trigeminal nerve denervation on the noncholinergic, nonadrenergic response to electrical transmural stimulation of the isolated rabbit iris sphincter muscle. The left ophthalmic nerve (first branch of the trigeminal nerve) was cut at the intracranial, peripheral site of the trigeminal ganglion and five to ten days later, the iris sphincter muscle isolated from the left eye (operated side) was found to produce a fast cholinergic contraction in response to electrical transmural stimulation and there was no evidence of noncholinergic, nonadrenergic contractions. On the other hand, in the iris sphincter muscle isolated from the right eye (control side), electrical transmural stimulation produced both cholinergic and noncholinergic, nonadrenergic contractile responses. Capsaicin and bradykinin produced noncholinergic, nonadrenergic contractile responses in the muscle from the control side, while in the iris sphincter from the trigeminally denervated eye there was no such response to application of these drugs. Exogenous substance P (SP) and carbachol produced a strong contractile response in both the trigeminally innervated and denervated sphincter muscles. Somatostatin, vasoactive intestinal polypeptide (VIP) and enkephalin were without effects. These observations suggest that the noncholinergic, nonadrenergic responses to electrical transmural stimulation are derived from the trigeminal nerve and that the mediator involved is probably SP or a related peptide.     

Evidence for two adenine derivative receptors in rat ileum which are not involved in the nonadrenergic, noncholinergic response.

The receptors mediating inhibition of the rat ileum by adenosine and adenine nucleotides were studied. ATP and ADP were more potent than AMP or adeonsine. Theophylline antagonized the effects of adenosine and AMP but not those of ATP or ADP. Preparations desensitized to ATP or ADP were still inhibited by adenosine and vice versa. The nonadrenergic, noncholinergic inhibition produced by field stimulation or nicotine was not attenuated by the presence of theophylline or desensitization to ATP. These data indicate that more than one adenine derivative receptor is present in rat ileum and that ATP and adenosine are unlikely candidates for the unknown transmitter.     

The effect of indomethacin and adenosine 5'-triphosphate on the excitatory innervation of the rate urinary bladder

Adenosine 5'-triphosphate (ATP) (20-400 microM) contracted 48% of isolated rat urinary bladder preparations but induced no response in the remainder. The response to ATP never exceeded 25% of the response to electrical stimulation in the presence of indomethacin (50 microM) plus hyoscine (25 microM) and usually developed more slowly than that to electrical stimulation. Autoinhibition could be produced to ATP by incubating the tissue with ATP (200 microM) for 20 min. Incubation of the tissue with ATP (200 microM) for 60 min in the presence of indomethacin (50 microM) and either hyoscine (25 microM) or hemicholinium-3 (500 microM) reduced but failed to abolish responses to electrical stimulation. Responses to acetylcholine were not affected by ATP (200 microM) in the presence of indomethacin and the output of acetylcholine induced by neuronal stimulation at 10 Hz was not inhibited by ATP (200 microM) or by indomethacin (50 microM). The results suggest a possible modulatory role for ATP in the excitatory innervation of the rat urinary bladder.     

Effects of arachidonic acid and indomethacin on the in vitro release of prostaglandins by aortic strips of spontaneously hypertensive rats

Aortic strips removed from spontaneously hypertensive (SH) rats and preincubated with arachidonic acid (1.0 X 10(-5) g/ml) for 15 min produced two times more prostaglandin (PG) like material than aortae unexposed to the precursor of PG biosynthesis. The stimulating effect of arachidonic acid was largely inhibited by indomethacin (1.0 X 10(-5) g/ml). Also, the release of PG-like material by aortic strips derived from SH rats treated with an intravenous injection of indomethacin (10 mg/kg) was inhibited by 74% compared with the control tissues. These results raised the possibility that the in vivo conversion of arachidonic acid by large arteries of SH rats may contribute to the hypotensive effect of this PG precursor in SH rats.     

Neural control of canine colon motor function: studies in vitro

The responses of strips of the canine colon to stimulation of intrinsic nerves and to the probable mediators of these nerves were studied in vitro. Studies were carried out using longitudinal and circular muscle strips from proximal and distal colon with field stimulation and addition of agents to the bath. Overall, these and other studies in vivo suggested that acetylcholine was an ubiquitous mediator of neural excitation. Norepinephrine had mixed inhibitory and excitatory effects, the latter only in circular muscle. Inhibitory effects of norepinephrine seemed to be both pre- and post-synaptic but no evidence that it was released by field stimulation was obtained. Substance P had excitatory effects chiefly by release of acetylcholine. It, in addition to norepinephrine, at least in circular muscle, deserves evaluation as the mediator of noncholinergic excitation to high frequency field stimulation. Although vasoactive intestinal peptide sometimes had inhibitory effects, these were incomplete and inconsistent. However, further evaluation of its possible role as a nonadrenergic, noncholinergic inhibitory mediator is required to determine if it is involved as one component in the response. Few qualitative differences existed between responses of various regions of the colon to potential neuromediators, although there were some consistent differences between responses of longitudinal and circular muscle. Some differences existed in responses obtained earlier in vivo and in vitro. In particular, inhibitory effects following excitation by substance P on field stimulation were found only in vivo. Nonadrenergic, noncholinergic inhibitory responses to field stimulation were consistently present only in vitro. These differences have not been explained.     

Neural control of relaxation in cat airways smooth muscle

Functional innervation of cat airways smooth muscle was examined in isolated segments of trachea and bronchi using electrical field stimulation (EFS) techniques. Field stimulation caused contraction in tissues at resting tone and biphasic responses (contraction followed by relaxation) in tissues precontracted with 5-hydroxytryptamine (5-HT). Contractions were abolished by 10(-6) M atropine. Inhibitory responses were dependent on impulse voltage, duration, and frequency. At low voltages (less than or equal to 10 V) and pulse durations (less than or equal to 0.3 ms), EFS induced relaxations were abolished by 3 X 10(-6) M tetrodotoxin (TTX). Greater stimulus parameters elicited TTX-resistant relaxations. Pretreatment of the tissues with 10(-6) M propranolol and 10(-5) M guanethidine caused rightward shifts in relaxation frequency-response curves. These findings indicate that cat airways are innervated by excitatory cholinergic, inhibitory adrenergic, and inhibitory nonadrenergic noncholinergic (NANC) nerves. Pretreatment of the tissues with hexamethonium, cimetidine, indomethacin, or nordihydroguaiaretic acid did not affect NANC relaxation responses. It is concluded that NANC inhibitory responses in cat airway smooth muscle are mediated through intrinsic postganglionic nerve fibers and occur independently of histamine H2-receptor activation and without involvement of cyclooxygenase or lipoxygenase products of arachidonic acid metabolism.     

Involvement of prostaglandins and histamine in radiation-induced temperature responses in rats

Exposure of rats to 1-15 Gy of gamma radiation induced hyperthermia, whereas exposure to 20-150 Gy produced hypothermia. Since radiation exposure induced the release of prostaglandins (PGs) and histamine, the role of PGs and histamine in radiation-induced temperature changes was examined. Radiation-induced hyper- and hypothermia were antagonized by pretreatment with indomethacin, a cyclooxygenase inhibitor. Intracerebroventricular administration of PGE2 and PGD2 induced hyper- and hypothermia, respectively. Administration of SC-19220, a specific PGE2 antagonist, attenuated PGE2- and radiation-induced hyperthermia, but it did not antagonize PGD2- or radiation-induced hypothermia. Consistent with an apparent role of histamine in hypothermia, administration of disodium cromoglycate (a mast cell stabilizer), mepyramine (H1-receptor antagonist), or cimetidine (H2-receptor antagonist) attenuated PGD2- and radiation-induced hypothermia. These results suggest that radiation-induced hyperthermia is mediated via PGE2 and that radiation-induced hypothermia is mediated by another PG, possibly PGD2, via histamine.     

The influence of prostaglandins on neurotransmission in the rabbit isolated vas deferens

Arachidonic acid and PGs of the D, E, F and I series were examined for influences on neurogenic contractions of the rabbit isolated vas deferens. This preparation exhibits two pharmacologically distinct contractions in response to electrical stimulation. All of the PGs tested inhibited the neurogenic contractions but the pattern of inhibition differed. PGE1 and PGI2 inhibited the adrenergic contractile phase more potently than the nonadrenergic, and PGF2 alpha exhibited the opposite selectivity. Arachidonic acid, PGE2 and PGD2 produced equipotent effects on both contractile phases, although PGE2 was the most potent in producing these effects. None of the PGs altered the concentration-response curve to norepinephrine. Contractile responses to ATP, a putative neurotransmitter, were inhibited by PGF2 alpha but not by the other PGs. These results suggest that the PG effects are predominantly prejunctional. The differing potencies of the PGs on the two neural components are consistent with the hypothesis that neurotransmitters in the vas deferens are released by distinct types of nerves.     

Nonneural components in the response of fresh human airways to electric field stimulation

Fresh human bronchi, obtained at thoracotomy and maintained at 37 degrees C, were studied in vitro to investigate their response to electric field stimulation (EFS). We found complex responses that were not only composed of a rapid initial nerve-mediated cholinergic contraction and a non-adrenergic nerve-mediated relaxation, but, in 80% of preparations, also of a tonic contraction with a sustained time course. This sustained phase was not blocked by the nervous conductance blocker tetrodotoxin (TTX) and was therefore not neurally mediated. Controlled transient cooling to 4 degrees C in the organ bath reduced this sustained phase selectively for several hours. The leukotriene (LT) antagonist FPL 55712, dexamethasone, which inhibits phospholipase A2, and the antiasthmatic drug cromolyn all reduced the sustained phase significantly. In 20% of strips, an additional TTX-resistant contraction was seen directly after the cholinergic phase. This contraction could be inhibited by indomethacin. A similar small peak sometimes appeared after selective blocking of either the cholinergic or the sustained phases. Experiments in which the epithelium was removed from the strips suggested that this indomethacin-sensitive response, but not the sustained phase, was dependent on the presence of epithelium. These results show that EFS of fresh human bronchi stimulated cholinergic and nonadrenergic inhibitory nerves and gave rise to a partly epithelium-dependent synthesis of arachidonic acid metabolites, which caused contractile responses that interfered with the neurally mediated responses.     

Activity of prostaglandin biosynthetic pathways in rat pancreatic islets

Isolated pancreatic islets of the rat were either prelabeled with [3H]arachidonic acid, or were incubated over the short term with the concomitant addition of radiolabeled arachidonic acid and a stimulatory concentration of glucose (17mM) for prostaglandin (PG) analysis. In prelabeled islets, radiolabel in 6-keto-PGF1 alpha, PGE2, and 15-keto-13,14-dihydro-PGF2 alpha increased in response to a 5 min glucose (17mM) challenge. In islets not prelabeled with arachidonic acid, label incorporation in 6-keto-PGF1 alpha increased, whereas label in PGE2 decreased during a 5 min glucose stimulation; after 30-45 min of glucose stimulation labeled PGE levels increased compared to control (2.8mM glucose) levels. Enhanced labelling of PGF2 alpha was not detected in glucose-stimulated islets prelabeled or not. Isotope dilution with endogenous arachidonic acid probably occurs early in the stimulus response in islets not prelabeled. D-Galactose (17mM) or 2-deoxyglucose (17mM) did not alter PG production. Indomethacin inhibited islet PG turnover and potentiated glucose-stimulated insulin release. Islets also converted the endoperoxide [3H]PGH2 to 6-keto-PGF1 alpha, PGF2 alpha, PGE2 and PGD2, in a time-dependent manner and in proportions similar to arachidonic acid-derived PGs. In dispersed islet cells, the calcium ionophore ionomycin, but not glucose, enhanced the production of labeled PGs from arachidonic acid. Insulin release paralleled PG production in dispersed cells, however, indomethacin did not inhibit ionomycin-stimulated insulin release, suggesting that PG synthesis was not required for secretion. In confirmation of islet PGI2 turnover indicated by 6-keto-PGF1 alpha production, islet cell PGI2-like products inhibited platelet aggregation induced by ADP. These results suggest that biosynthesis of specific PGs early in the glucose secretion response may play a modulatory role in islet hormone secretion, and that different pools of cellular arachidonic acid may contribute to PG biosynthesis in the microenvironment of the islet.     

Prostaglandins do not modulate the positive chronotropic and inotropic effects of sympathetic nerve stimulation and injected norepinephrine in the isolated blood perfused canine atrium

In isolated canine atrium, perfused with blood from a donor dog, the infusions of both prostaglandins (PG)I₂ and E₂ (0.1–1 μg/min) into the sinus node arterial cannula neither altered the sinus rate and developed tension nor the positive chronotropic and inotropic responses elicited by either electrical stimulation or by injected norepinephrine. Infusion of arachidonic acid (10–100 μg/min), a precursor of PGs, or indomethacin (15–20 μg/min), an inhibitor of PG synthesis, into the sinus node arterial cannula also failed to alter the increase in sinus rate or developed tension produced by either adrenergic stimulus in the isolated atria. When arachidonic acid, 100–300 μg/kg or PGI₂, 1 μg/kg, were injected into the jugular vein of the donor dog, they produced a fall in systemic blood pressure; this effect of arachidonic acid but not of PGI₂ was abolished by indomethacin, 1 mg/kg. During administration of either arachidonic acid or indomethacin to the donor dog, the positive chronotripic and inotropic responses to adrenergic stimuli in the isolated atria also remained unaltered. These data indicate that PGs do not modulate adrenergic transmission in the blood perfused canine atrium.     

Stimulation frequency-dependent nonadrenergic noncholinergic airway responses of the guinea pig

We examined the inhibitory and excitatory components of the nonadrenergic noncholinergic (NANC) innervation of the guinea pig airways by in vivo and in vitro methods. Electrical stimulation of the vagus in chloralose-urethan-anesthetized guinea pigs after cholinergic and adrenergic blockade produced peripheral airway constriction (insufflation pressure) and tracheal relaxation (pouch pressure). Vagal stimulation was applied for 90 s at 5-V pulses of 2-ms duration at frequencies of 5, 15, 25, and 35 Hz in each group (n = 6). The pouch relaxation peaked at 15 Hz. The insufflation pressure was highest at 5 Hz. Field stimulations of the same frequencies were applied on tracheal spirals and lung parenchymal strips. The maximal relaxation of the trachea occurred at 15-35 Hz. The lung parenchymal strip tensions increased almost linearly as the frequency increased from 5 to 35 Hz. The results of the study indicated a frequency-dependent response for both excitatory and inhibitory components of the NANC, which operate at different frequencies for optimal responses.     

Effect of trifluoperazine, a calmodulin antagonist, on prostaglandin output from the guinea-pig uterus

Trifluoperazine, a calmodulin antagonist, inhibited the A23187-induced increase in outputs of prostaglandin (PG) F-2 alpha and 6-oxo-PGF-1 alpha from the Day 7 and Day 15 guinea-pig uterus superfused in vitro. The basal outputs of, and the arachidonic acid-induced increase in outputs of PGF-2 alpha, PGE-2 and 6-oxo-PGF-1 alpha from the guinea-pig uterus were not inhibited by trifluoperazine. In contrast, indomethacin inhibited A23187-stimulated, arachidonic acid-stimulated and the basal outputs of PGs from the guinea-pig uterus, indicating that trifluoperazine was not inhibiting cyclo-oxygenase. Since the action of A23187 is dependent upon extracellular Ca2+, the present findings provide evidence that calmodulin is involved in Ca2+-induced increases in uterine PG output from the guinea-pig uterus. Trifluoperazine, but not indomethacin, inhibited A23187-induced contraction of the guinea-pig uterus, which is consistent with calmodulin being involved in smooth muscle contraction. Arachidonic acid treatment did not contract the guinea-pig uterus. These findings indicate that PGs are not involved in the contraction induced by A23187. Other findings of interest were (i) trifluoperazine caused a small, sometimes significant (P less than 0.05), increase in uterine PG output, (ii) exogenous arachidonic acid failed to increase PGF-2 alpha output from the Day 15 uterus in contrast to the stimulant action of A23187, and (iii) exogenous arachidonic acid caused a fairly large increase in uterine PGE-2 output in contrast to the small effect with A23187.     

Pharmacology of scorpion (Lychas laevifrons Pock) venom with special reference to its neuromuscular activity

L. laevifrons venom caused irreversible blockade of electrically induced twitch responses on phrenic nerve diaphragm and chick biventer cervicis preparation. The venom lowered cat blood pressure, caused a brief cardiac arrest and increased cutaneous capillary permeability. It contracted several smooth muscle preparations. The quick contraction produced on guinea pig ileum was partly antagonized by mepyramine and completely by methysergide. The residual slow contraction was antagonized by SC 19220, a prostaglandin blocker. Haemolysis was not produced by the venom on human RBC. LD50 of crude venom in mice was 13.8 mg/kg (iv).     

Glycerylprostaglandin synthesis by resident peritoneal macrophages in response to a zymosan stimulus

Cyclooxygenase (COX)-2 oxygenates arachidonic acid (AA) and 2-arachidonylglycerol (2-AG) to endoperoxides, which are subsequently transformed to prostaglandins (PGs) and glycerylprostaglandins (PG-Gs). PG-G formation has not been demonstrated in intact cells treated with a physiological agonist. Resident peritoneal macrophages, which express COX-1, were pretreated with lipopolysaccharide to induce COX-2. Addition of zymosan caused release of 2-AG and production of the glyceryl esters of PGE2 and PGI2 over 60 min. The total quantity of PG-Gs (16 +/- 6 pmol/10(7) cells) was much lower than that of the corresponding PGs produced from AA (21,000 +/- 7,000 pmol/10(7) cells). The differences in PG-G and PG production were partially explained by differences in the amounts of 2-AG and AA released in response to zymosan. The selective COX-2 inhibitor, SC236, reduced PG-G and PG production by 49 and 17%, respectively, indicating a significant role for COX-1 in PG-G and especially PG synthesis. Time course studies indicated that COX-2-dependent oxygenation rapidly declined 20 min after zymosan addition. When exogenous 2-AG was added to macrophages, a substantial portion was hydrolyzed to AA and converted to PGs; 1 microm 2-AG yielded 820 +/- 200 pmol of PGs/10(7) cells and 78 +/- 41 pmol of PG-Gs/10(7) cells. SC236 reduced PG-G and PG production from exogenous 2-AG by 88 and 76%, respectively, indicating a more significant role for COX-2 in the utilization of exogenous substrate. In conclusion, lipopolysaccharide-pretreated macrophages produce PG-Gs from endogenous 2-AG during zymosan phagocytosis, but PG-G formation is limited by substrate hydrolysis and inactivation of COX-2.     

Neural control of canine colon motor function: studies in vivo

The responses of the circular muscle of canine colon to stimulation of intrinsic nerves and to the probable mediators of these nerves were studied in vivo. In vivo studies were carried out using close intra-arterial injections and local field stimulation of proximal, mid-, and distal colon while recording circumferential contractions. Our results suggest that acetylcholine is the major excitatory mediator, but another excitatory mediator could be released by high frequency field stimulation after atropine. Norepinephrine had mixed inhibitory and excitatory effects, but no evidence was obtained that it was released by field stimulation. Substance P had mainly excitatory effects partly by a mechanism involving nerves and partly by a direct effect on muscle; it in addition to norepinephrine deserves further evaluation as the mediator of noncholinergic excitation to high frequency field stimulation. There is no explanation of the inhibition it produced after initial excitation during field stimulation. Vasoactive intestinal peptide had inhibitory effects but these were incomplete and inconsistent. This may be related to our inability to demonstrate relaxation or inhibition to field stimulation after atropine. Further evaluation of the possible role of vasoactive intestinal peptide and other agents as nonadrenergic, noncholinergic inhibitory mediators is required.     

Possible involvement of adenine nucleotides in the neurotransmission of the mouse urinary bladder

The urinary bladder of the mouse contracts to several agonists, namely acetylcholine, noradrenaline, adrenaline, histamine, angiotensin, serotonin, purine nucleotides and prostaglandin F2 alpha. Atropine partially reduced the contraction induced by electrical stimulation, whereas propranolol and tolazoline were ineffective. The atropine resistant component of the neurogenic response was reduced by indomethacin. Methysergide and diphenhydramine were ineffective. Desensitization of the bladder by alpha,beta-methylene ATP abolished the response to ATP and greatly reduced the non-adrenergic non-cholinergic component of the neurogenic response. The results suggest that ATP could be the transmitter responsible for the non-cholinergic non-adrenergic contraction of the mouse urinary bladder.