P1-purinoceptor-mediated modulation of neural noradrenaline and ATP release in guinea-pig vas deferens

BACKGROUND/AIMS: Mutations in p53, a tumor suppressor gene located on chromosome 17p, are the most frequent genetic alterations found in human cancers. Increased intracellular concentration of p53, which is frequently but not systematically related to p53 mutation, has been proposed to be associated with poor prognosis in some tumor types. In colorectal cancer, this significance is still a matter of debate. To directly investigate the relationship between prognosis and p53 mutation, this study screened a series of 85 colorectal carcinomas for mutations in exons 5-8 of this gene. METHODS: Polymerase chain reaction-amplified products from tumor DNA were analyzed by denaturing gradient gel electrophoresis and direct DNA sequencing. RESULTS: Forty-four tumors were found to be mutated (52%). A strong correlation between the presence of a mutation and short survival was observed (P = 0.003). When tumors were classified according to their histological stage, a multivariate Cox model analysis showed that p53 mutation, rather than 17p allelic loss (previously proposed to convey prognostic information), was retained as the only independent prognostic factor (relative risk, 2.25; 95% confidence interval, 1.06-4.80; P < 0.029). CONCLUSIONS: Combined with staging, direct monitoring of p53 mutation improves prognostic accuracy for colorectal cancer.     

An analysis of the postjunctional component of denervation supersensitivity in the isolated vas deferens of the guinea-pig

An attempt was made to devise evidences for a role of calcium ions in the postjunctional component of denervation supersensitivity. The evidences obtained are: chronic postganglionic denervation increases sensitivity and maximum response of the vas deferens to cumulative concentrations of calcium and alters the pattern of response to low-calcium, potassium-rich Krebs solution; denervation supersensitivity could not be demonstrated after depolarization, and in KC1-Ringer or in Ca2+/--free Krebs solution the rate of loss of responsiveness to acetylcholine was delayed after denervation whereas the rate of loss of responsiveness to noradrenaline was unaffected. It is suggested that the postjunctional component of denervation supersensitivity in the isolated vas deferens of the guinea-pig is due, at least partially, to an increased availability of a membrane-bound calcium store(s) associated to an enhanced cell membrane permeability to the ion.     

Sympathetic efferent pathways projecting to the vas deferens

The abdominal and pelvic sympathetic nervous system controlling the vas deferens has elaborate mechanisms to preserve its function against various injuries. The main sympathetic signals to the vas deferens proceed the common pathway in mammalians, which consists of the lumbar splanchnic nerve, caudal mesenteric plexus, hypogastric nerve, pelvic plexus and its branches. On the way of this common pathway, some signals cross to the other side of the body at the level of the caudal mesenteric plexus and/or the pelvic plexus. The preganglionic axons passing through the hypogastric nerve very likely provide a bilateral innervation to postganglionic neurons in the pelvic plexuses, which also exhibit crossing to the bilateral vasa deferentia. The sympathetic nerves originating from the thoracic spinal cord are of minor importance in contraction of the vas deferens but possibly influence it by the hormonal system consisting of the major splanchnic nerve and the adrenal medulla. When the common pathway is interrupted, various compensatory mechanisms are generated: enhancement of the remaining sympathetic pathways or reorganization of synaptic connection in the pelvic plexus. Surgical reconstruction of the transected hypogastric nerve is possible and cross-innervation mechanism via the hypogastric nerve can also be preserved. Elevation of intraluminal pressure at the cauda epididymis/proximal vas deferens induced by nerve impulse pushes the spermatozoa out to the ampulla and distention of the wall of the ampulla triggers its contraction to emit the content into the urethra. After seminal emission, a portion of the seminal fluid remaining in the vas deferens moves in a retrograde direction to the cauda epididymis for the next emission. It remains to be seen whether similar mechanisms in animals are at work in humans.     

A1 adenosine receptor modulation of electrically-evoked contractions in the bisected vas deferens and cauda epididymis of the guinea-pig

1. The effects of adenosine receptor agonists upon both electrically-evoked and phenylephrine-induced contractile responses were investigated in the bisected vas deferens and the cauda epididymis of the guinea-pig. Electrical field-stimulation (10 s trains of pulses at 9 Hz, 0.1 ms duration, supramaximal voltage) elicited biphasic and monophasic contractile responses from preparations of bisected vas deferens and cauda epididymis, respectively; these responses were abolished by tetrodotoxin (300 nM). 2. In the prostatic half of the vas deferens the A1 selective adenosine receptor agonists, N6-cyclopentyladenosine (CPA) and (2S)-N6-[2-endo-norbornyl]adenosine ((S)-ENBA) and the non-selective A1/A2 adenosine receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA) inhibited electrically-evoked contractions (pIC50+/-s.e.mean values 6.15+/-0.24, 5.99+/-0.26 and 5.51+/-0.24, respectively). The responses to CPA were blocked by the A1 adenosine receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine, DPCPX (100 nM). 3. In the epididymal half of the vas deferens NECA potentiated (at < or = 100 nM) and inhibited (at > or = 1 microM) electrically-evoked contractions. In the presence of the non-selective alpha-adrenoceptor antagonist phentolamine (3 microM), the alpha1-adrenoceptor antagonist, prazosin (100 nM), or at a reduced train length (3 s) NECA inhibited electrically-evoked contractions (pIC50 values 6.05+/-0.25, 5.97+/-0.29 and 5.71 +/-0.27, respectively). CPA (at 10 microM) also inhibited electrically-evoked contractions in this half of the vas deferens. In the presence of prazosin (100 nM), CPA also inhibited electrically-evoked contractions (pIC50 6.14+/-0.67); this effect was antagonized by DPCPX (30 nM, apparent pK(B) 8.26+/-0.88). In the presence of the P2 purinoceptor antagonist, suramin (300 microM), CPA (up to 1 microM) potentiated electrically-evoked contractions. 4. NECA, CPA and APNEA potentiated electrically-evoked contractions in preparations of cauda epididymis (pEC50 values 7.49+/-0.62, 7.65+/-0.74 and 5.84+/-0.86, respectively), the response to CPA was competitively antagonized by DPCPX (100 nM) with an apparent pK(B) value of 7.64+/-0.64. 5. The alpha1-adrenoceptor agonist phenylephrine elicited concentration-dependent contractile responses from preparations of bisected vas deferens and cauda epididymis. NECA (1 microM) potentiated responses to phenylephrine (< or = 1 microM) in the epididymal, but not in the prostatic half of the vas deferens. In preparations of epididymis NECA (1 microM) shifted phenylephrine concentration response curves to the left (4.6 fold). In the presence of a fixed concentration of phenylephrine (1 microM), NECA elicited concentration-dependent contractions of preparations of the epididymal half of the vas deferens and of the epididymis (pEC50 values 7.57+/-0.54 and 8.08+/-0.18, respectively). NECA did not potentiate responses to ATP in either the epididymal half of the vas deferens or the epididymis. 6. These studies are consistent with the action of stable adenosine analogues at prejunctional A1 and postjunctional A1-like adenosine receptors. The prejunctional A1 adenosine receptors only inhibit the electrically-evoked contractions of purinergic origin (an effect predominant in the prostatic half of the vas deferens). At the epididymis, where electrically-evoked contractions are entirely adrenergic, the predominant adenosine receptor agonist effect is a potentiation of alpha1-adrenoceptor-, but not of ATP-induced contractility.     

Effects of suramin on contractions of the guinea-pig vas deferens induced by analogues of adenosine 5'-triphosphate

1. Adenosine 5'-triphosphate (ATP) and some of its analogues contract the guinea-pig vas deferens, acting via receptors which have been classified as P2X-purinoceptors. We have recently shown, however, that the effects of ATP are enhanced, rather than inhibited, by the non-selective P2 antagonist, suramin, and that this enhancement could not easily be explained in terms of inhibition by suramin of the breakdown of ATP. We therefore investigated the effects of suramin on contractions induced by ATP analogues, to define the structure-activity relationships of the suramin-resistant response. 2. In the absence of suramin, the order of potency for ATP analogues was adenosine 5'-(alpha,beta-methylene)triphosphonate (AMPCPP) = P1,P5-diadenosine pentaphosphate (Ap5A) = adenosine 5'-tetraphosphate (Ap4) > adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) = adenylyl 5'-(beta,gamma-methylene) diphosphonate (AMPPCP) > P1,P5-diadenosine tetraphosphate (Ap4A) > adenosine 5'-O-(2- thiodiphosphate) (ADP beta S) > 2-methylthioadenosine 5'-triphosphate (MeSATP) > or = ATP > adenosine 5'-diphosphate (ADP). This is generally in agreement with previously reported structure-activity relationships in this tissue. 3. In the presence of suramin (1 mM), responses to Ap5A, Ap4A, AMPPCP, ADP beta S and ADP were abolished or greatly reduced, and contractions induced by AMPCPP, Ap4 and ATP gamma S were inhibited. Contractions induced by MeSATP however, like those induced by ATP itself, were not reduced, but at concentrations above 100 microM were enhanced. In the presence of suramin (1 mM) the order of potency of analogues was therefore AMPCPP = Ap4> ATP = MeSATP> ATP gamma S, with all other analogues tested being essentially inactive at concentrations up to 500 microM.4. Contractile responses of the vas deferens to transmural nerve stimulation (1-50 Hz) in the presence of the alpha-adrenoceptor antagonist, phentolamine (10 microM), were abolished by suramin (1 mM). This is in agreement with previous reports that suramin inhibits the excitatory junction potential, a response thought to be mediated by P2 purinoceptors. It is however hard to reconcile the evidence implicating ATP as the non-adrenergic transmitter responsible for this response with the failure of suramin to inhibit the contractions induced by ATP itself while abolishing nerve-mediated contractions.5. In conclusion, these results confirm our previous findings of a suramin-resistant component to the ATP-induced contraction in the guinea-pig vas deferens, and show that the structure-activity relationships of this response are not identical to those of any known P2-purinoceptor subclass. Although the inhibition by suramin of the breakdown of ATP may contribute to the suramin-resistance of some of the ATP analogues, it does not appear to provide the full explanation.     

Inhibition of purinergic transmission by prostaglandin E1 and E2 in the guinea-pig vas deferens: an electrophysiological study

1. The effects of prostaglandin E1 (PGE1) and E2 (PGE2) on postjunctional electrical activity in the guinea-pig vas deferens evoked by sympathetic nerve stimulation were investigated using both intracellular and focal extracellular recording techniques in vitro. 2. Bath application of PGE1 (1-100 nM) or PGE2 (0.1-100 nM) concentration-dependently inhibited the amplitudes of all excitatory junction potentials (e.j.ps) evoked during short trains of stimuli (10 stimuli at 1 Hz). Increasing the duration of nerve stimulation (100 stimuli at 1 Hz) did not overcome this inhibitory effect. At these concentrations PGE1 and PGE2 were without any apparent inhibitory effect on the amplitudes of spontaneous e.j.ps. 3. Local application of PGE1 (10-100 nM) or PGE2 (10-30 nM) markedly reduced the frequency of occurrence of excitatory junction currents (e.j.cs) evoked by trains of 20-100 stimuli at 1 to 4 Hz without changing the amplitudes of spontaneous e.j.cs or the configuration of the nerve terminal impulse. 4. In the presence of PGE1 or PGE2, raising the frequency of stimulation (from 1 to 4 Hz), increased the likelihood of e.j.c. occurrence. 5. The postjunctional electrical activity recorded in the guinea-pig vas deferens is believed to be due to ATP released from the sympathetic nerve endings. Thus the present study demonstrates that both PGE1 and PGE2 powerfully inhibit quantal ATP release in the guinea-pig vas deferens.     

Studies on the neurophysiology of the vas deferens

In order to investigate the mechanism of sperm transport along the genital ducts, intraluminal pressure of isolated segments of the vas deferens was recorded in vivo. Responses to filling and mechanical as well as pharmacologic and electric stimulation of the autonomic nervous system were monitored. Contraction waves were initated in response to the stretch of filling and from mechanical stimulation. Pharmacologic response was variable. Low doses of alpha-adrenergic stimulant produced an increase in frequency of the contraction wave. Large doses of the same drug induced stroking contraction of the entire vas. alpha-Blocking drugs did not alter the rhythmic activity of the vas. beta-Adrenergic stimulation blocked peristaltic activity while administration of parasympathomimetic drugs increased the force of contraction. Electric stimulation of the hypogastric nerve produced strong sustained contractions. These data suggest that, whenever stretched, the vas deferens responds by regular peristaltic waves of low amplitude. These peristaltic waves can be enhanced by sympathomimetics or electric stimulation of the sympathetic system. The contents of the vas are propulsed into the urethra through strong rhythmic contractions of the entire vas.     

Opposite modulation of noradrenaline and ATP release in guinea-pig vas deferens through prejunctional beta-adrenoceptors: evidence for the beta 2 subtype

Activation of prejunctional beta-adrenoceptors has been suggested to increase the release of noradrenaline but to decrease the neural release of ATP in the guinea-pig vas deferens. Experiments were carried out to determine the subtype of beta-adrenoceptor involved. In [3H]-noradrenaline-preincubated tissues superfused with medium containing prazosin and suramin, isoprenaline (1-100 nM), salbutamol (0.01-1 microM) and terbutaline (0.1-10 microM) increased the overflow of tritium but reduced the overflow of ATP elicited by electrical stimulation (210 pulses/7 Hz). The effects of isoprenaline were blocked by the beta 2-selective antagonist 1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3- [(1-methylethyl)amino]-2-butanol (ICI 118,551; 100 nM). In prazosin- and suramin-free medium, isoprenaline (100 nM) did not change the overflow of ATP elicited by exogenous noradrenaline (10 microM). Isoprenaline (1-100 nM), salbutamol (0.01-1 microM) and terbutaline (0.1-10 microM) reduced the initial twitch contraction elicited by electrical stimulation (210 pulses/7 Hz) in prazosin- and suramin-free medium as well as the isolated purinergic neurogenic contraction obtained by exposure to prazosin. They increased or tended to increase the secondary sustained concentration elicited by electrical stimulation in prazosin- and suramin-free medium as well as the isolated adrenergic neurogenic contraction obtained in the presence of suramin. The inhibition by isoprenaline of the isolated purinergic contraction was attenuated by ICI 118,551 (100 nM) but not by the beta 1-selective antagonist 1-[2-((3-carbamoyl- 4-hydroxy)phenoxy)ethylamino]-3-[4-(1-methyl-4- trifluoromethyl-2-imidazolyl)phenoxy]-2-propanol (CGP 20712A; 100 nM). The results confirm the opposite beta-adrenoceptor-mediated modulation of noradrenaline and neural ATP release in the guinea-pig vas deferens. They show that the prejunctional beta-adrenoceptor is of the beta 2-subtype.     

Dopamine receptors and dopaminergic nerves in the vas deferens of the rat

Phentolamine antagonized competitively the effects of noradrenaline (pA2 = 7.1), dopamine (pA2 = 8.0) and tyramine (pA2 = 8.2). Haloperidol had a pA2 value of 7.3 against dopamine and 6.5 against noradrenaline. Apomorphine antagonized competitively dopamine (pA 2 = 4.8) and tyramine (pA2 = 5.1) and noncompetitively antagonized noradrenaline (pD'2 = 3.6). From these data it is concluded that these antagonists interact with dopamine receptors and alpha-adrenergic receptors. Apomorphine (10-4 M) attenuated the maximal response to dopamine and field stimulation, whereas the same concentration of apomorphine potentiated the maximal response to noradrenaline. Assuming that tyramine and field stimulation release the naturally occurring neurohumoral transmitter from adrenergic nerve endings, it is concluded that dopamine is the physiologically functional neurohumoral transmitter in the rat vas deferens which, when released, stimulates specific dopamine receptors.     

Azoospermia due to aperistalsis of the vas deferens: successful treatment with pseudoephedrine

Three patients presenting with infertility were found to have low volume azoospermia. All 3 were taking sympatholytic medications; 2 were taking antipsychotics and 1 was taking an alpha-blocker. Low volume azoospermia may result from the use of sympatholytic medications, which cause aperistalsis of the adrenergically innervated vas deferens and seminal vesicles. Two patients had normal spermatogenesis on biopsy and were unobstructed on vasography. In 1 patient, biopsy and vasography were avoided. Pseudoephedrine, a sympathomimetic agent, was given to all 3 patients, resulting in marked improvement in semen analysis parameters. A trial of pseudoephedrine can obviate the need for biopsy and vasography in such patients.     

Somatostatin-induced inhibition of neurotransmission in the mouse isolated vas deferens is resistant to pertussis toxin

The potential effects of pertussis toxin pretreatment on the inhibitory effect of somatostatin (SRIF) and the selective SRIF receptor agonist, seglitide, were studied in mouse vas deferens and these were compared with its effect on the negative chronotropic action of carbachol in mouse atria. Somatostatin and seglitide caused a concentration-dependent inhibition of neurogenically mediated contractile responses in the vas deferens (EC50 values of 15 and 0.6 nM respectively). There was no difference in their potencies in preparations removed from pertussis toxin pretreated mice. In contrast, the negative chronotropic action of carbachol in mouse atria was abolished by pretreatment with pertussis toxin. We conclude that, in contrast to muscarinic receptor activation in mouse atria, the inhibitory effect of somatostatin in the vas deferens is not mediated by a pertussis toxin sensitive G-protein. The high potency of seglitide suggests that the SRIF receptor involved is of the SRIF1 type.     

Tetrodotoxin-sensitive action potentials in smooth muscle of mouse vas deferens

Action potentials were recorded during impalements of some but not all smooth muscle cells of mouse vas deferens in response to both nerve stimulation and intracellular current injection. They were resistant to blockade by nifedipine (0.1-1.0 microM) but were blocked by tetrodotoxin (TTX, 0.2-1.0 microM) when this was added in the presence of nifedipine. It is suggested that voltage-dependent sodium (Na+) channels are present in mouse vas deferens that function to amplify calcium (Ca2+) influx through voltage-dependent Ca2+ channels.     

Active transport of 3H-bretylium in the rat vas deferens in vitro

A total of 409 Staphylococcus aureus strains were classified with 17 typing sera into 16 groups and 14 types. Strains with the formula a b+ c e and h+ i k l were most frequently met with. The majority of strains isolated from typical staphylococcal diseases contained antigens e', h+ and/or h++ whereas those associated with atypical infections showed antigens b++ and o most frequently. Classification by Oeding and Williams' scheme yielded a less wide variety of serological units: most strains fell into group 4; spontaneously agglutinating and group 1, 2 and 3 strains were next in order.     

Analysis of spermatozoa from the proximal vas deferens of vasectomized men

As the health care system is oriented to provide service with finite dollars, nursing educators are being asked to demonstrate how continuing education for staff improves patient outcomes. The purpose of this study was to evaluate the impact of an orthopedic-geriatric continuing education program for nurses on the elderly patient who had sustained a hip fracture. A significant difference was found between the control and experimental unit patients with respect to time to first ambulation and length of stay on the orthopedic unit.     

Study of TRH in the postganglionic sypnasis of the isolated human vas deferens

Cummulative dose-response curves to l-noradrenaline and in presence of different concentrations of TRH (10(-3), 10(-4), 10(-5), 10(-6), 10(-7), and 10(-8) M) were investigated. 25 segments of vas deferens (escrotal tract) were obtained from different surgical patients of urological disorders. At the lower concentration (greater than 10(-7) M), we can not appreciate any influence of TRH on the l-noradrenaline response. Nevertheless there was a reduction of response to l-noradrenaline, with displacement of the curves to the right side, for the high concentrations, and a proportional ratio to the concentration to TRH.