Alpha,beta-methylene Adenosine Triphosphate Research Articles

Investigation of the role of adrenergic and non‐nitrergic, non‐adrenergic neurotransmission in the sheep isolated internal anal sphincter

Nitric oxide is widely established as an important neurotransmitter in the control of anal sphincter tone; although, a number of other transmitters have also been tentatively implicated. Whilst alpha-adrenoceptor antagonists reduce anal sphincter pressure in man, the role of noradrenaline as a possible transmitter is poorly characterised. We have investigated the contribution of these transmitters to neurogenic relaxations, and evaluated the possible role of a non-nitrergic, non-adrenergic transmitter. The magnitude and duration of neurogenic responses were examined by measuring responses to electrical field stimulation (EFS) in segments of sheep internal anal sphincter following the development of spontaneous myogenic tone. Neurogenic relaxations induced by EFS were significantly reduced in the presence of N(G)-nitro-L-arginine methyl ester (L-NAME) suggesting major involvement of nitric oxide as a neurotransmitter. The duration of neurogenic relaxations was inversely related to the frequency of EFS, with contractile responses often manifest at higher frequencies. The duration of relaxations at high frequencies of EFS was increased by bretylium (adrenergic neurone blocker) and prazosin (alpha(1)-adrenoceptor antagonist). At higher frequencies of EFS, 60% of preparations also produced a residual non-nitrergic, non-adrenergic, apamin-sensitive relaxation which was unaffected by vasoactive intestinal polypeptide (VIP) and inhibitors of purinergic responses [suramin, pyridoxal-phosphate-6-azophenyl 2',4' disulfonic acid (PPADS) and alpha,beta-methylene adenosine triphosphate (ATP)]. However, MRS2179 (P2Y(1) receptor antagonist) showed a modest inhibitory effect. We conclude that endogenous noradrenaline acts via postjunctional alpha(1)-adrenoceptors to antagonize neurogenic relaxations that are largely mediated by nitric oxide. Our results indicate the involvement of a non-nitrergic, non-adrenergic, apamin-sensitive transmitter which is inhibited by MRS2179, suggesting a possible role for purines.

Effects of in utero bladder outflow obstruction on fetal sheep detrusor contractility, compliance and innervation.

Congenital bladder outflow obstruction caused by posterior urethral valves is a common cause of end stage renal failure in boys. We hypothesized that fetal bladder outflow obstruction perturbs detrusor contractility and innervation and bladder storage volume-pressure relationships. Severe bladder outflow obstruction was induced in male fetal sheep by placing a urethral ring and urachal ligation midway through gestation at 75 days. Fetuses were examined 30 days after surgery, when urinary tract dilatation, enlarged bladders and histologically abnormal kidneys were documented. Isolated strips of bladder detrusor from sham operated and obstructed fetuses were subjected to electrical field stimulation, carbachol, KCl and alpha-beta methylene-adenosine triphosphate. Whole bladder storage characteristics were determined by filling cystometry and bladder innervation was investigated by immunohistochemistry and Western blot. Tension-frequency contractility studies showed that obstructed fetal bladder strips were significantly hypocontractile versus sham operated controls in response to electrical field stimulation and the specific agonists carbachol, KCl and alpha-beta methylene-adenosine triphosphate. Hypocontractility was greater with nerve mediated stimulation than with carbachol, suggesting relative denervation. Reduced innervation was confirmed by S100 and protein gene product 9.5 immunohistochemistry and by measuring a significant reduction in protein gene product 9.5 protein expression using Western blot. Filling cystometry showed that obstructed fetal bladders appeared more compliant (Delta V/Delta P, where Delta V is the change in volume and Delta P is the change in pressure) with larger capacity, more flaccidity and yet retained stress relaxation. In response to severe experimental fetal bladder outflow obstruction the bladder becomes large and hypocontractile, and has aberrant innervation.

Mechanisms of excitatory transmission in circular smooth muscles of the guinea pig seminal vesicle.

Cellular mechanisms of excitatory neuromuscular transmission in circular smooth muscles of the seminal vesicle were investigated. Circular smooth muscles of the seminal vesicle of the guinea pig were isolated. Changes in membrane potential produced by transmural nerve stimulation were recorded using intracellular microelectrode techniques. Changes in the intracellular Ca ion concentration induced by transmural nerve stimulation were measured in preparations loaded with Ca indicator fura-PE3. Responses produced by bath applied norepinephrine and alpha,beta-methylene adenosine triphosphate (ATP) were also examined. Transmural nerve stimulation evoked excitatory junction potentials that triggered action potentials and also caused transient increases in [Ca2+] (Ca transients). Nifedipine abolished action potentials, leaving underlying excitatory junction potentials unchanged, and reduced the amplitude of Ca transients. Excitatory junction potentials were blocked by alpha,beta-methylene ATP or guanethidine but not by phentolamine. A train of transmural nerve stimulation evoked oscillatory changes in membrane potential and [Ca2+], which were abolished by phentolamine or inhibited by nifedipine. Nifedipine insensitive components were abolished by cyclopiazonic acid. Norepinephrine depolarized the membrane and elicited oscillatory potentials with an associated elevation in [Ca2+]. These responses were inhibited by nifedipine and abolished by additional application of cyclopiazonic acid. Transient depolarization with an associated increase in [Ca2+] was elicited by alpha,beta-methylene ATP and [Ca2+] responses but no potential changes were inhibited by nifedipine. Circular smooth muscles of the guinea pig seminal vesicle receive a projection of sympathetic nerves that release norepinephrine to initiate slow depolarization through the activation of alpha-adrenoceptors. These nerves also release ATP to elicit excitatory junction potentials. Neurally released norepinephrine and ATP are increased [Ca2+] by the influx of Ca2+ through L-type Ca2+ channels and also by the release of Ca2+ from internal stores.

Adenosine 5'-triphosphate (ATP) is an excitatory cotransmitter with noradrenaline to the smooth muscle of the rat prostate gland.

1. This study investigated whether adenosine 5'-triphosphate (ATP) is involved in neurotransmission to the rat prostate gland. 2. Fluorescence immunohistochemistry carried out on formaldehyde-fixed and frozen sections of rat prostate showed immunoreactivity for the P2X(1)-receptor in the fibromuscular stroma surrounding the secretory acini but not in the glandular epithelium. P2X(2)-, P2X(3)-, P2X(4)- and P2X(7)-receptors were immunonegative in the rat prostate stroma. Double-staining procedures showed P2X(1)-receptor immunoreactivity to be colocalized with alpha-actin immunoreactivity. 3. Isolated organ bath studies investigated whether drugs, which modify purinergic mechanisms, are able to affect contractility of the rat prostate gland. Suramin (100 micro M) and alphabetamethylene ATP (10 micro M) inhibited contractile responses to trains of electrical-field stimulation (70 V, 0.5 ms, 0.1-2 Hz) in the absence and presence of prazosin (300 nM). Responses to 5-20 Hz were unaffected by suramin or alphabetamethylene ATP. 4. Exogenous application of ATP analogues to unstimulated isolated preparations of rat prostate produced concentration-dependent suramin (100 micro M) sensitive transient contractions with a relative order of potency: alphabetamethylene ATP>betagammamethylene ATP>ATP. 5. Adenosine and adenosine 5'-monophosphate (AMP) did not produce contractile responses. 6. These results suggest that P2X(1)-receptors for ATP, which mediate contractions are present in the fibromuscular stroma of the rat prostate. The relative order of potency of ATP analogues in producing contractions of the rat prostate is consistent with the activation of P2X(1)-receptors. Inhibition by suramin and alphabetamethylene ATP of electrically evoked nerve-mediated contractions of the rat prostate implies that ATP contributes to this contractile response and is therefore a cotransmitter with noradrenaline during low-frequency stimulation.

Mechanisms of Excitatory Transmission in Circular Smooth Muscles of the Guinea pig Seminal Vesicle

Cellular mechanisms of excitatory neuromuscular transmission in circular smooth muscles of the seminal vesicle were investigated. Circular smooth muscles of the seminal vesicle of the guinea pig were isolated. Changes in membrane potential produced by transmural nerve stimulation were recorded using intracellular microelectrode techniques. Changes in the intracellular Ca ion concentration induced by transmural nerve stimulation were measured in preparations loaded with Ca indicator fura-PE3. Responses produced by bath applied norepinephrine and alpha,beta-methylene adenosine triphosphate (ATP) were also examined. Transmural nerve stimulation evoked excitatory junction potentials that triggered action potentials and also caused transient increases in [Ca2+] (Ca transients). Nifedipine abolished action potentials, leaving underlying excitatory junction potentials unchanged, and reduced the amplitude of Ca transients. Excitatory junction potentials were blocked by alpha,beta-methylene ATP or guanethidine but not by phentolamine. A train of transmural nerve stimulation evoked oscillatory changes in membrane potential and [Ca2+], which were abolished by phentolamine or inhibited by nifedipine. Nifedipine insensitive components were abolished by cyclopiazonic acid. Norepinephrine depolarized the membrane and elicited oscillatory potentials with an associated elevation in [Ca2+]. These responses were inhibited by nifedipine and abolished by additional application of cyclopiazonic acid. Transient depolarization with an associated increase in [Ca2+] was elicited by alpha,beta-methylene ATP and [Ca2+] responses but no potential changes were inhibited by nifedipine. Circular smooth muscles of the guinea pig seminal vesicle receive a projection of sympathetic nerves that release norepinephrine to initiate slow depolarization through the activation of alpha-adrenoceptors. These nerves also release ATP to elicit excitatory junction potentials. Neurally released norepinephrine and ATP are increased [Ca2+] by the influx of Ca2+ through L-type Ca2+ channels and also by the release of Ca2+ from internal stores.

Effects of In Utero Bladder Outflow Obstruction on Fetal Sheep Detrusor Contractility, Compliance and Innervation

Congenital bladder outflow obstruction caused by posterior urethral valves is a common cause of end stage renal failure in boys. We hypothesized that fetal bladder outflow obstruction perturbs detrusor contractility and innervation and bladder storage volume-pressure relationships. Severe bladder outflow obstruction was induced in male fetal sheep by placing a urethral ring and urachal ligation midway through gestation at 75 days. Fetuses were examined 30 days after surgery, when urinary tract dilatation, enlarged bladders and histologically abnormal kidneys were documented. Isolated strips of bladder detrusor from sham operated and obstructed fetuses were subjected to electrical field stimulation, carbachol, KCl and alpha-beta methylene-adenosine triphosphate. Whole bladder storage characteristics were determined by filling cystometry and bladder innervation was investigated by immunohistochemistry and Western blot. Tension-frequency contractility studies showed that obstructed fetal bladder strips were significantly hypocontractile versus sham operated controls in response to electrical field stimulation and the specific agonists carbachol, KCl and alpha-beta methylene-adenosine triphosphate. Hypocontractility was greater with nerve mediated stimulation than with carbachol, suggesting relative denervation. Reduced innervation was confirmed by S100 and protein gene product 9.5 immunohistochemistry and by measuring a significant reduction in protein gene product 9.5 protein expression using Western blot. Filling cystometry showed that obstructed fetal bladders appeared more compliant (Delta V/Delta P, where Delta V is the change in volume and Delta P is the change in pressure) with larger capacity, more flaccidity and yet retained stress relaxation. In response to severe experimental fetal bladder outflow obstruction the bladder becomes large and hypocontractile, and has aberrant innervation.

P2X purinoceptors in postmortem human cerebral arteries.

Pharmacological studies have demonstrated that various purinoceptors are involved in the control of the cerebral vascular tone in many species. In this study, the existence of P2X purinoceptors in the postmortem human cerebral arteries was investigated with organ-bath pharmacology, autoradiography, and immunohistochemistry. Specimens were obtained from the M2 region of the middle cerebral arteries from human cadavers with an age range of 53-91 years and postmortem time of 37-54 h. Application of alpha,beta-methylene adenosine triphosphate (ATP) produced concentration-dependent contraction in the arterial ring, whereas transmural nerve stimulation and noradrenaline did not elicit contraction. Autoradiography using [3H]alpha,beta-methylene ATP (a radioligand for P2X purinoceptors) showed specific [3H]alpha,beta-methylene ATP binding sites in the smooth-muscle cells of the postmortem human cerebral arteries. Immunohistochemistry with specific P2X1 purinoceptor antibodies revealed positive staining exclusively in the smooth muscle of the same specimens. All these results demonstrate the existence of P2X purinoceptors in human cerebral arteries, which were still functionally active despite the long postmortem time. The results from this study suggest that the postmortem human cerebral arteries can be useful specimens for studying the P2X purinoceptor-mediated responses.

An electrophysiological study of developmental changes in the innervation of the guinea-pig taenia coli.

A sucrose-gap technique was used to study the development of neuromuscular transmission in the taenia coli of fetal, 1- to 2-day-old, 3- to 4-week-old and 3-month-old guinea-pigs. In addition, the effects of exogenous, alpha,beta-methylene adenosine 5'-triphosphate (ATP), noradrenaline, vasoactive intestinal polypeptide (VIP) and carbachol were examined. Taking the response to a single pulse of electrical field stimulation as the index of a developed neuromuscular junction, it was apparent that the non-adrenergic inhibitory system arose before, and matured more quickly than, the cholinergic system. The inhibitory system was present by 8 weeks of gestation in some fetuses, but, in contrast, excitatory cholinergic transmission was not seen until birth. As evidenced by responses to carbachol, alpha,beta-methylene ATP and VIP, cholinergic, purinergic and VIP receptors were present on the smooth muscle at the earliest ages studied. No changes in sensitivity to these agents were noted throughout development, although in adults the level of the maximum responses increased.

Characteristics of the vagally driven non-adrenergic, non-cholinergic inhibitory innervation of ferret gastric corpus.

This paper reports a quantitative in vivo study on the vagal activation of the intramural non-adrenergic, non-cholinergic inhibitory nerves in the ferret gastric corpus. The nature of the inhibitory neurotransmitter was also investigated. In the atropinized, guanethidine-treated, urethane-anaesthetized ferret, electrical stimulation (10 s at 20 V, 1-20 Hz, 0.5 ms pulses) of the cervical vagi produced a prompt fall in intracorpus pressure that was related to the stimulus frequency. The maximal response was achieved at 10 Hz. The time taken for the intracorpus pressure to return to pre-stimulus levels after a 10 s period of stimulation was related to the stimulus frequency; at 10 Hz the pressure took approximately 11 min to recover. In contrast to studies in the cat (Martinson & Muren, 1963), there was no detectable difference in the electrical threshold for activation of the vagal excitatory and vagal inhibitory fibres. The nature of the vagal non-adrenergic, non-cholinergic inhibitory neurotransmitter was investigated using a variety of antagonists and agonists. Adenosine triphosphate (ATP), adenosine, alpha beta-methylene ATP and beta gamma-methylene ATP all contracted the corpus in the presence of vagotomy, atropine, guanethidine and indomethacin. The vagally induced fall in corpus pressure was not blocked by high doses of alpha beta-methylene ATP. A variety of peptides were investigated for their effects on corpus pressure in the presence of atropine, guanethidine and vagotomy. Bombesin, pentagastrin, substance P, cholecystokinin octapeptide (CCK-8) and bradykinin all produced an increase in intracorpus pressure. Neurotensin and vasoactive intestinal polypeptide (VIP) both decreased intracorpus pressure, and of the two VIP most closely mimicked the response to vagal activation of the non-cholinergic, non-adrenergic inhibitory neurones. The results provide support for the involvement of a peptide (possibly VIP) rather than a purine in the vagally driven decrease in intracorpus pressure in the ferret.