Activation of neural cholecystokinin-1 receptors induces relaxation of the isolated rat duodenum which is reduced by nitric oxide synthase inhibitors.

Cholecystokinin (CCK) influences gastrointestinal motility, by acting on central and peripheral receptors. The aim of the present study was to determine whether CCK has any effect on isolated duodenum longitudinal muscle activity and to characterize the mechanisms involved. Isolated segments of the rat proximal duodenum were mounted for the recording of isometric contractions of longitudinal muscle in the presence of atropine and guanethidine. CCK-8S (EC50: 39; 95% CI: 4.1-152 nM) and cerulein (EC50: 58; 95% CI: 18-281 nM) induced a concentration-dependent and tetrodotoxin-sensitive relaxation. Nomeganitro-L-arginine (L-NOARG) reduced CCK-8S- and cerulein-induced relaxation (IC50: 5.2; 95% CI: 2.5-18 microM) in a concentration-dependent manner. The magnitude of 300 nM CCK-8S-induced relaxation was reduced by 100 microM L-NOARG from 73 +/- 5.1 to 19 +/- 3.5% in an L-arginine but not D-arginine preventable manner. The CCK-1 receptor antagonists proglumide, lorglumide and devazepide, but not the CCK-2 receptor antagonist L-365,260, antagonized CCK-8S-induced relaxation in a concentration-dependent manner. These findings suggest that CCK-8S and cerulein activate intrinsic nitrergic nerves acting on CCK-1 receptors in order to cause relaxation of the rat duodenum longitudinal muscle.

Differential effects of estrogen and antiestrogen on transforming growth factor gene expression in endometrial adenocarcinoma cells.

While antiestrogens are useful agents in the treatment of breast cancer, the usefulness of these agents in the treatment of endometrial cancer remains controversial. There is some concern that the currently available antiestrogens may have partial agonist activity in uterine tissue. To better understand the mechanisms by which estrogens and antiestrogens modulate growth of endometrial adenocarcinoma cells, we have compared the effects of 17-beta estradiol and three antiestrogens, 4-hydroxytamoxifen (OH-TAM), ICI 164384, and LY 117018 on proliferation and transforming growth factor (TGF) mRNA accumulation in two human endometrial adenocarcinoma cell lines. In HEC-50 cells, neither estradiol nor anti-estrogens had any effect on cell proliferation or TGF mRNA abundance under estrogen-depleted culture conditions [basal medium containing 1% twice charcoal-treated fetal bovine serum (ctFBS)] or in the presence of estrogen (basal medium containing 5% fetal bovine serum). At very high concentrations, both estradiol and OH-TAM caused a small decrease in HEC-50 cell proliferation in medium containing 5% serum. In contrast, the antiestrogens had different effects on Ishikawa cells, depending upon the culture conditions. In medium containing 5% fetal bovine serum, the antiestrogens inhibited cell proliferation and significantly decreased TGF-alpha mRNA abundance and TGF-alpha secretion. OH-TAM was more potent than the other antiestrogens. Under these culture conditions, estradiol had no effect on cell proliferation or TGF-alpha mRNA levels but increased TGF-alpha secretion. In medium supplemented with 1% ctFBS, estradiol increased cell proliferation and TGF-alpha mRNA (2.72-fold, P less than 0.005) and TGF-alpha secretion (700 +/- 156 versus 250 +/- 23 pg/10(6) cells/24 h, P less than 0.05), whereas OH-TAM, which also stimulated cell proliferation, reduced TGF-alpha mRNA abundance (P less than 0.05) but had no significant effect on TGF-alpha secretion. Under these conditions, ICI 164384 and LY 117018 had no effect on either cell proliferation or TGF-alpha expression. Estradiol treatment decreased, whereas OH-TAM increased, epidermal growth factor receptors in Ishikawa cells. Both estradiol and the antiestrogens decreased TGF-beta 1 mRNA abundance when cells were grown in media containing 1% ctFBS. In summary, the response of human endometrial adenocarcinoma cells to estrogen and antiestrogens varied between cell lines and was dependent upon the culture conditions used. In addition, OH-TAM, unlike the other two antiestrogens tested, had growth-stimulating effects on Ishikawa cells.

Transforming growth factor gene expression in human endometrial adenocarcinoma cells: regulation by progestins.

In an attempt to understand the antiproliferative effects of progestins in endometrial cancer, we have examined the effects of the potent progestin, medroxyprogesterone acetate (MPA), on the cell proliferation and the expression of transforming growth factor (TGF) alpha and beta genes in human endometrial adenocarcinoma cell lines. The two cell lines used were Ishikawa, var 1, and HEC-50. In addition, the effects of exogenous TGF-alpha and anti-epidermal growth factor (EGF) receptor monoclonal antibody on cell proliferation were determined. Incubation of both cell lines with MPA resulted in a time- and dose-dependent inhibition of cell proliferation. Half-maximal growth inhibition was observed at 0.6 nM. In Ishikawa cells, the relative abundance of TGF-alpha was significantly reduced by MPA. A significant decrease in TGF-alpha mRNA was apparent 6 h after exposure to MPA and a further decrease was seen 12-24 h after addition of the progestin. The concentration of TGF-alpha immunoreactivity in conditioned medium of MPA-treated cells was also significantly reduced compared to control cultures. MPA had no effect on TGF-alpha expression by HEC-50 cells. EGF mRNA was not detected by Northern blot analysis in either cell type. MPA had no significant effect on EGF receptor mRNA abundance but resulted in a small increase in EGF receptor number in Ishikawa cells. Anti-EGF receptor monoclonal antibody (0.6-6 nM) inhibited Ishikawa cell growth but had no effect on HEC-50 cell proliferation. Exogenous TGF-alpha stimulated proliferation of both cell lines, but Ishikawa cells were significantly more sensitive to exogenous TGF-alpha than HEC-50 cells. Furthermore, TGF-alpha could reverse the growth inhibitory effects of MPA on Ishikawa cells. A decrease in TGF-beta mRNA abundance was also observed in MPA-treated Ishikawa and HEC-50 cells. This effect was of small magnitude, variable, and only observed after prolonged exposure to MPA. These observations are consistent with the hypothesis that the antiproliferative effects of progestins on Ishikawa cells are mediated by decreased expression and autocrine action of TGF-alpha. Since similar growth inhibition is also seen in the HEC-50 cells in which progestins have no effect on TGF-alpha expression, additional mechanisms are likely to be involved in the antiproliferative effects of progestins in human endometrial cancer.

Identification and characterization of a rat decidual insulin-like growth factor-binding protein complementary DNA.

A partial cDNA which encodes the rat homolog of human placental protein-12, the low mol wt insulin-like growth factor-binding protein (IGFBP-1), has been isolated from a rat decidual cDNA library using low stringency hybridization with a human IGFBP-1 cDNA probe. The incomplete cDNA obtained from this library was used to screen a rat liver cDNA library from which a full-length cDNA was obtained. The predicted amino acid sequence of rat IGFBP-1 showed 66%, 29%, and 34% sequence identity with the human IGFBP-1, the human GH-dependent binding protein IGFBP-3, and rat IGFBP-2, the BP secreted by buffalo rat liver cells, respectively. The rat IGFBP-1 cDNA hybridized with a 1.6-kilobase transcript which was easily detected in uterine RNA from the pseudopregnant rat and RNA from the liver and kidney of adult rats. A low level of expression was apparent in the brain and the diestrous uterus. Of the tissues examined the order of abundance of IGFBP-1 mRNA was deciduoma tissue greater than or equal to liver much greater than kidney much greater than uterus greater than brain. The 1.6-kb mRNA was more abundant in RNA from neonatal rat liver than in that from maternal liver, but was not detected in total RNA (50 micrograms) from other neonatal rat tissues (kidney, lung, brain, and heart). Under stringent conditions, rat IGFBP-1 did not hybridize with RNA from the BRL-3A rat liver cell line. In food-deprived rats, hepatic IGFBP-1 mRNA was increased 10.0 +/- 2.2-fold compared to that in control rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Phorbol esters differentially regulate the expression of insulin-like growth factor-binding proteins in endometrial carcinoma cells.

We have examined the effects of protein kinase-C (PKC) activation on expression of the six known insulin-like growth factor-binding proteins (IGFBPs) by human endometrial carcinoma cells. Each of six known IGFBPs was expressed in one or more of the three cell lines examined. The addition of 10(-7) M 4 beta-phorbol 12-myristate 13-acetate (PMA) to HEC-50 and HEC-1B cells resulted in changes in cell morphology, growth inhibition, activation of PKC, and an increase in expression of IGFBP-1. PMA had no effect on these parameters in the Ishikawa cell line, which did not express IGFBP-1. In HEC-50 cells, the effect of PMA was blocked by the concomitant addition of the PKC inhibitor staurosporin and the simultaneous addition of cycloheximide. PMA also resulted in an increase in IGFBP-3 in HEC-50 cells and an increase in IGFBP-6 expression in HEC-1B cells. In contrast, IGFBP-3 expression was down-regulated by PMA in HEC-1B and Ishikawa cells. The abundance of IGFBP-2 and IGFBP-5 mRNAs was also reduced in HEC-1B and Ishikawa cells, respectively. IGFBP-4 was expressed only in HEC-50 cells and was not affected by PMA treatment. These data establish a role for the PKC pathway in regulation of expression of IGFBP-1, -2, -3, and -5 in endometrial adenocarcinoma cells and illustrate the complexity of cell type-specific expression of the IGFBPs.

Nitric oxide blunts sympathetic response of pregnant normotensive and hypertensive rat arteries.

Rat pregnancy is associated with a blunted response to vasocontrictors both in vivo and in vitro as well as a decrease in arterial pressure. We examined the influence of pregnancy on neurally induced vasoconstrictor and vasodilator responses of the isolated mesenteric arterial bed from normotensive Wistar and spontaneously hypertensive nonpregnant and 20-day pregnant rats and determined the possible role of nitric oxide (NO) in modulating these responses. MAP (mm Hg) in pregnant normotensive (98+/-1, n=13) and hypertensive (136+/-5, n=13) rats was lower (P<.05) than in nonpregnant controls (114+/-2, n=14, and 174+/-3, n=12, respectively). In isolated mesenteric arterial beds, electrical field stimulation (EFS; 34 V, 3 ms, 10-64 Hz) of perivascular nerves at basal tone induced a frequency-dependent increase in perfusion pressure that was significantly (P<.001) greater in preparations from hypertensive compared with normotensive rats. Pregnancy was associated with a significant decrease in the maximal vasoconstrictor response elicited by EFS in both normotensive and hypertensive groups compared with their nonpregnant controls. In phenylephrine-preconstricted mesenteric beds, EFS (60 V, 1 ms, 1-8 Hz) elicited a similar frequency-dependent decrease in perfusion pressure in normotensive and hypertensive groups, but pregnancy did not influence these responses. In the presence of the NO synthase inhibitor N(omega)-nitro-L-arginine (200 micromol/L), the maximal vasoconstrictor response induced by EFS was significantly (P<.001) augmented in both normotensive and hypertensive groups, and the differences observed between pregnant and nonpregnant groups were abolished. Responses to sodium nitroprusside were not affected by pregnancy, although they were greater in preparations from hypertensive rats. These results indicate that NO contributes to pregnancy-associated diminished vasoconstrictor response to sympathetic stimulation in the mesenteric arterial bed of both normotensive and hypertensive rats.

Insulin binding to human ovaries.

Human ovarian tissue samples were obtained at the time of laparotomy, and plasma membrane fractions were prepared and used in receptor assays. Incubations of the membrane fraction were performed with [125I]insulin (porcine), and Scatchard analysis of binding showed biphasic curves. The high affinity sites had an average concentration of 57.4 +/- 7.9 (+/- SEM) fmol/mg protein and a dissociation constant of 3.5 +/- 0.9 nM (n = 9). Neither affinity nor number of binding sites changed significantly during the menstrual cycle. We conclude that there is high affinity binding of [125I]insulin to human ovarian plasma membranes.

Blockers of the L-arginine-nitric oxide-cyclic GMP pathway facilitate baroreceptor resetting.

We investigated the role of nitric oxide on rapid (25- and 40-minute) baroreceptor resetting during the onset of acute hypertension in rats treated with NG-nitro-L-arginine, an inhibitor of nitric oxide synthesis, and methylene blue, an inhibitor of guanylate cyclase. The effect of treatment with glibenclamide, an ATP-dependent K+ channel blocker, was also investigated. Arterial hypertension was provoked in a ramp progression by the drug NG-nitro-L-arginine alone or in association with aortic coarctation. Whole aortic nerve activity and carotid pressure were recorded in the anesthetized rats. The extent of rapid resetting was evaluated by means of the ratio (delta Systolic Threshold Pressure/delta Control Diastolic Pressure) x 100 as well as by the extent of displacement of the pressure-nerve activity curve defined by the ratio (delta Mean Arterial Pressure at 50% of maximum activity/delta Mean Arterial Pressure) x 100. All groups gave the same increase in mean arterial pressure at 25 and 40 minutes after the onset of hypertension. A greater extent of resetting to hypertensive levels was observed in the treated groups compared with coarctation alone. At 40 minutes after the onset of hypertension, the coarctation and nitro-L-arginine groups exhibited a further increase in the extent of resetting. The rats submitted to glibenclamide plus coarctation presented a slight but significant decrease in gain. These findings suggest that an active L-arginine-nitric oxide-cyclic GMP pathway blunts rapid resetting during the onset of hypertension. In addition, they also indicate that ATP-dependent K+ channels can also modulate rapid resetting of the baroreceptors to hypertensive levels.

Heart rate variability and baroreceptor function in chronic diabetic rats.

In conscious chronic (12 to 18 weeks) streptozotocin diabetic rats, we examined the changes in basal heart rate, with particular attention to heart rate variability assessed by evaluating the standard deviation (bpm) of the lengths of adjacent pulse pressure. We also investigated in anesthetized rats the ability of the aortic baroreceptors to acutely (30 minutes) reset to hypertensive levels. For this purpose, pressure-nerve activity curves for the baroreceptors were obtained, and gain (slope of the curve) and mean arterial pressure at 50% of maximal baroreceptor activity were calculated. The shift of the pressure-nerve activity curve was used as an index of resetting. Conscious diabetic rats (n=6) exhibited lower mean arterial pressure (93+/-6 versus 109+/-4 mm Hg), heart rate (272+/-25 versus 359+/-11 bpm), and heart rate variability (18+/-7 versus 36+/-6 bpm) than control rats (n=7). Under anesthesia, diabetic rats (n=7) and control rats (n=8) exhibited similar mean arterial pressure (113+/-6 versus 109+/-7 mm Hg in control rats ), mean arterial pressure at 50% of maximal baroreceptor activity (117+/-5 versus 107+/-6 bpm), gain (1.66+/-0.08 versus 1.81+/-0.05%/mm Hg), and extent of resetting (44+/-12 versus 49+/-9%) to hypertensive levels. The present study demonstrated that conscious chronic diabetic rats presented lower heart rate variability than control rats. On the other hand, chronic diabetes was not associated with alterations in baroreceptor function or its ability to rapidly reset to hypertensive levels.

Power spectra of arterial pressure and heart rate in streptozotocin-induced diabetes in rats.

BACKGROUND: Chronic diabetes is associated with alterations in autonomic modulation of the cardiovascular system. Although the rat has been used extensively in studies of experimental diabetes, there have been no reports on the changes in autonomic modulation of the cardiovascular function in chronic diabetic rats. OBJECTIVE: To examine chronic diabetic rats to determine the autonomic modulation of arterial pressure and heart rate variabilities in the time and frequency domain. MATERIALS AND METHODS: Diabetes was induced in rats by a single injection of streptozotocin, and 30 min of pulsatile arterial pressure was recorded in conscious rats, 5, 10-20 days and 12-18 weeks after the streptozotocin injection. Control rats were injected with vehicle. Beat-by-beat systolic arterial pressure and heart rate were obtained from pulsatile pressure. The spectral density powers of systolic arterial pressure and heart rate were calculated using fast Fourier transformation, and integrated in low-(0.015-0.25 Hz), mid- (0.25-0.75 Hz) and high- (0.75-3.0 Hz) frequency bands. The standard deviations of systolic arterial pressure and heart rate were also calculated. RESULTS: Basal systolic arterial pressure and heart rate were reduced in diabetic animals studied 10-20 days and 12-18 weeks after the streptozotocin injection. The standard deviations of systolic arterial pressure and heart rate were also reduced in the chronically diabetic animals. Diabetes reduced low- and mid-frequency variability but not the high-frequency variability of systolic arterial pressure. The low-frequency variability, but not the mid-frequency variability, of the heart rate was also reduced, while the high-frequency variability of the heart rate was reduced in the more chronically diabetic rats. CONCLUSION: Our findings that the mid-frequency band variability of arterial pressure was reduced in diabetic patients suggest that sympathetic modulation of the cardiovascular system is impaired, corroborating other studies in such patients using this and other approaches.

Role of spinal nitric oxide synthase-dependent processes in the initiation of the micturition hyperreflexia associated with cyclophosphamide-induced cystitis.

The aim of this study was to examine the participation of nitrergic neurotransmission in the initiation of micturition hyperreflexia associated to cyclophosphamide (CP)-induced cystitis in rats. Micturition threshold volume was significantly reduced 4 h after CP administration (100 mg/kg, i.p.); this reduction was attenuated by intra-arterially injected N(G)-nitro-l-arginine-methyl ester (l-NAME), a non selective nitric oxide synthase (NOS) inhibitor, but not by intravesical infusion of S-methyl-l-thiocitrulline (l-SMTC), another structurally different NOS inhibitor. Interestingly, l-NAME failed to affect micturition threshold volume in normal rats. The magnitude of isolated detrusor strips contractions elicited by either carbachol or nerve activation was significantly reduced in CP-treated rats but was unaffected by the addition of N(G)-nitro-l-arginine (l-NOARG), a nonselective NOS inhibitor. In contrast, intrathecal l-NAME and l-SMTC but not N(G)-nitro-d-arginine-methyl ester (d-NAME) administration augmented the micturition threshold volume in CP-treated rats in an l-arginine preventable manner. As with the systemic injection, intrathecal l-NAME also did not affect the micturition threshold volume in normal rats. Four hours after CP injection, the number of neuronal NOS immunoreactive or nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) positive neurons in spinal lumbosacral segments (L6-S2) was not altered whereas the number of c-Fos immunoreactive neurons increased significantly in the dorsal gray commissural nucleus (DGC), the parasympathetic sacral nucleus (PSN) and lamina X of these segments. Ca(2+)-dependent, but not Ca(2+)-independent NOS activity increased significantly in spinal L6-S2 segments but not in thoracic segments of CP-treated rats. These data indicate that the micturition hyperreflexia observed in the initial hours of CP-induced cystitis is associated with an increase in Ca(2+)-dependent NOS activity in spinal L6-S2 segments suggesting an increased production of nitric oxide (NO). The increased production of NO in these spinal segments appears to be necessary for the initiation of the micturition hyperreflexia.

In vitro effects of calcium entry blockers, chlorpromazine and fenoterol upon human pregnant myometrium contractility.

The inhibitory effects of nifedipine, verapamil, cinnarizine (calcium entry blockers), chlorpromazine (a putative calmodulin antagonist) and fenoterol (a beta 2-adrenoceptor agonist) on contractility in human isolated pregnant myometrium were studied. Spontaneous contractions (present in 93% of the preparations) were inhibited in a concentration-related manner by these compounds in the following order of potency: nifedipine greater than verapamil much greater than cinnarizine greater than chlorpromazine. Cinnarizine was effective only at a concentration greater than 100 microM. Fenoterol, at 10 microM, did not produce an inhibitory effect but decreased the frequency of spontaneous contractions. All drugs, except fenoterol, produced a concentration-dependent relaxation of K+-induced contractions in the following order of sensitivity: nifedipine greater than verapamil much greater than chlorpromazine. Cinnarizine produced only about 40% of relaxation. Under these conditions nifedipine and verapamil were about 80 and 5 fold more potent respectively than when tested against spontaneous contractions. The potencies of chlorpromazine and cinnarizine did not differ in the two experimental conditions. Both the spontaneous and K+-induced contractions were inhibited in a time-dependent manner in Ca2+-free media and the responses were almost completely abolished in 70-100 min. Calcium addition to the medium rapidly restored both spontaneous or K+-induced contractions. To investigate further the role of intracellular calcium, K+-depolarized preparations contracted by calcium 3 mM (40-60% of maximal contractions) were relaxed by these compounds. Nifedipine and verapamil showed a relaxation time course similar to that induced by calcium removal. Cinnarizine and fenoterol had no relaxant effect while chlorpromazine induced a slight and slow relaxation. 6 These findings suggest that calcium influx and calmodulin are involved in spontaneous contractions of pregnant human myometrium in vitro. Since nifedipine and verapamil were more potent against K+-induced than spontaneous contractions, calcium channels activated by these conditions could be different. Finally, fenoterol, a beta 2-adrenoceptor agonist, widely used as a tocolytic agent, blocked neither spontaneous nor K+-induced contractions.

Mechanism of neuromuscular blockade induced by phenthonium, a quaternary derivative of (-)-hyoscyamine, in skeletal muscles.

1. The mechanisms underlying the postjunctional blockade induced by phenthonium [N-(4-phenyl) phenacyl 1-hyoscyamine] were investigated in mammalian and amphibian muscles. This muscarinic antagonist was previously shown to enhance specifically the spontaneous acetylcholine (ACh) release at concentrations that blocked neuromuscular transmission. 2. In both rat diaphragm and frog sartorius muscles, phenthonium (Phen, 1-100 microM) depressed the muscle twitches elicited by nerve stimulation (IC50: 23 microM and 5 microM, respectively), and blocked the nerve-evoked muscle action potential. The neuromuscular blockade was not reversed after incubation with neostigmine. 3. Equal concentrations of Phen decreased the rate of rise and prolonged the falling phase of the directly elicited action potential in frog sartorius muscle fibres, indicating that the drug also affects the sodium and potassium conductance. 4. Phen (50 and 100 microM) protected the ACh receptor against alpha-bungarotoxin (BUTX) blockade in the mouse diaphragm allowing recording of endplate potentials and action potentials after 5 h wash with physiological salt solution. 5. Phen (10-100 microM) produced a concentration- and voltage-dependent decrease of the endplate current (e.p.c.), and induced nonlinearity of the current-voltage relationship. At high concentrations Phen also shortened the decay time constant of e.p.c (tau(e.p.c.)) and reduced its voltage sensitivity. 6. At the same range of concentrations, Phen also reduced the initial rate of [125I]-BUTX binding to junctional ACh receptors of the rat diaphragm (apparent dissociation constant = 24 microM), the relationship between the degree of inhibition and antagonist concentration being that expected for a competitive mechanism. 7. It is concluded that Phen affects the electrical excitability of the muscle fibre membrane, and blocks neuromuscular transmission through a mechanism that affects the agonist binding to its recognition site and ionic channel conductance of the nicotinic ACh receptor.

Anxiolytic effect of nitric oxide synthase inhibitors microinjected into the dorsal central grey.

Nitric oxide synthase (NOS) accounts for most of the NADPH-diaphorase neuronal activity in the brain. NADPH-diaphorase-positive neurones have been localized at the dorso-lateral part of the periaqueductal grey (PAG), a region related to anxiety. Microinjections of the NOS inhibitors L-NAME (10-200 nmol 0.5 microliter-1) and L-NOARG (10-100 nmol) at this site induced anxiolytic-like effects in the elevated plus maze. These effects, however, occurred only at a limited range of doses and the dose-effect curve had a bell shape, higher doses of both compounds tending to be anxiogenic. The anxiolytic effect of L-NAME was antagonized by a previous microinjection of L-arginine (50 nmol 0.5 microliter-1). These results suggest that NO may play a role in PAG neurotransmission involved in anxiety.

Oxytocin releases atrial natriuretic peptide from rat atria in vitro that exerts negative inotropic and chronotropic action.

Our previous experiments suggested that natriuresis induced by blood volume expansion, was brought about by oxytocin (OT)-stimulated atrial natriuretic peptide (ANP) release from the right atrium. We hypothesized that the ANP released might exert effects on the atrium itself and therefore carried out in vitro experiments to test this hypothesis. Heart rate and isometric tension were recorded from isolated rat atria mounted in an organ bath. Oxytocin exerted a dose-related, negative chrono- and inotropic effect with a minimal effective concentration (MEC) of 3 microM, 10-fold higher than required for ANP to exert comparable effects. The effects of OT were not blocked by atropine suggesting that they were not mediated via release of acetylcholine. Eight-bromoguanosine 3'-5'-cyclic monophosphate (cGMP) had similar effects to those of OT and ANP, suggesting that the effects of ANP were mediated by cGMP. When isolated ventricles, left or right atria, were incubated in vitro, OT had a dose-related effect to stimulate the release of ANP into the medium only from right atria with a MEC of 0.1 microM. A specific OT antagonist, F792 (1 microM), inhibited basal release of ANP and blocked the stimulatory action of OT on ANP release. The results support the hypothesis that OT, acting on its putative receptors in the right atrium, stimulates the release of ANP which then exerts a negative chrono- and inotropic effect via activation of guanylyl cyclase and release of cGMP. The ability of the oxytocin antagonist to reduce basal release of ANP from atria incubated in vitro supports the hypothesis that these effects could be physiologically significant. We hypothesize that blood volume expansion via baroreceptor input to the brain causes the release of OT which circulates to the heart and stimulates the release of ANP from the right atrium. This ANP then has a negative ino- and chronotropic effect in the atrium and possibly a negative inotropic effect in the right ventricle, left atrium and left ventricle, to produce an acute reduction in cardiac output that, coupled with its peripheral vasodilating actions, causes a rapid reduction in effective circulating blood volume. The ANP released would also act on the kidneys to cause natriuresis and ANP acts within the brain to inhibit water and salt intake leading to a gradual recovery of circulating blood volume to normal.

In vitro and in vivo effects of nitric oxide synthase inhibitors and nitric oxide inactivators on the South American opossum ileocolonic junction.

The potential role of nitrergic nerves in the regulation of the South American (SA) opossum ileocolonic junction (ICJ) function was investigated. In vitro, the effects of nitric oxide (NO) synthase inhibitors and NO inactivators on the non-adrenergic non-cholinergic (NANC) nerve-mediated relaxations of the circular muscle of the SA opossum ICJ were determined by employing isolated strips. Electrical field stimulation (0.2-8.0 Hz) caused frequency-dependent NANC relaxations. Nicotine and ATP also induced concentration dependent NANC relaxations that were abolished by tetrodotoxin (TTX). The relaxation response induced by NANC nerve activation was reduced in a dose dependent manner by NO synthase inhibitors while vasoactive intestinal peptide (VIP) and sodium nitroprusside (SNP) induced relaxations were uninfluenced by these drugs. In vivo, the NO synthase inhibitor, L-NAME, administered into the local artery caused a raise in intraluminal pressure of the ICJ in anaesthetized SA opossums in a L-arginine-preventable manner. Hydroquinone and pyrogallol, while being able to reduce, in a superoxide dimutase (SOD) reversible manner, the relaxations induced by exogenous NO failed to affect the NANC nerve-induced relaxations. Finally, neurones and nerve fibres in the myenteric plexus as well as varicose nerve fibres on the circular smooth layer were positive for NADPH-diaphorase activity. These findings indicate that nitrergic nerves inhibit ICJ circular smooth muscle in vitro and in vivo but cast doubts on the neuromediator being the NO radical.

Rat duodenum nitrergic-induced relaxations are cGMP-independent and apamin-sensitive.

The effects of the K+ channel blockers, apamin, tetraethylammonium and 4-aminopyridine, upon the relaxations of the isolated rat proximal duodenum induced by nitregic nerve activation, nitric oxide (NO), the NO donor 3-morpholinosydnonimine (SIN-1) and Br-cyclic GMP were determined. The effects of the guanylate cyclase inhibitors, cystamine and N-methylhydroxylamine, on NO-, SIN-1- and nitrergic nerve-induced responses were also investigated. Apamin inhibited nitrergic nerve-, NO-and SIN-1-induced relaxations but did not affect those induced by Br-cGMP. Tetraethylammonium and 4-aminopyridine as well as cystamine and N-methylhydroxylamine failed to affect the relaxations caused by any of the agents tested. These findings indicate that, in the rat proximal duodenum, nitrergic nerve activation as well as exogenous nitric oxide cause relaxation through a cGMP-independent, apamin sensitive mechanism.

Pharmacological profile of nitrergic nerve-, nitric oxide-, nitrosoglutathione- and hydroxylamine-induced relaxations of the rat duodenum.

Activation of inhibitory nonadrenergic noncholinergic (NANC) nerves in the rat duodenum cause relaxations, which are reduced by nitric oxide synthase (NOS) inhibitors indicating that this response involves a nitrergic neurotransmission. The precise nature of the nitrergic neurotransmitter is still controversial since nitric oxide (NO) scavengers and superoxide generators, even in the presence of superoxide dismutase inhibitors, failed to inhibit nitrergic neurotransmission mediated relaxations. In order to understand the role of NOS in nitrergic neurotransmission and considering that N-OH-arginine (OH-L-Arg), L-citrulline, NO, S-nitrosoglutathione (GSNO) and hydroxylamine (NH2OH) can be formed in cells during the N(G)-oxidation of L-arginine catalyzed by NOS we explored whether any of these products could exhibit biological properties comparable to those of the nitrergic neurotransmitter. After establishing which of them was able to relax the rat duodenum, the pharmacological profile of such effect was determined employing oxyhemoglobin (OxyHb), pyrogallol (PYR), hydroquinone (HQ), hydroxocobalamin (HC) or carboxy-PTIO (C-PTIO) and compared with that of nerve mediated relaxations. NO, GSNO and NH2OH, but not OH-L-ARG and L-citrulline, caused concentration-dependent relaxations that were not affected by tetrodotoxin or L-NOARG. OxyHb almost abolished NO-induced relaxations but decreased only marginally the magnitude of nerve-, NH2OH- and SNG-induced relaxations. PYR, HQ and C-PTIO reduced significantly GSNO- and NO- induced relaxations but did not affect those induced by NH2OH or nerve activation. In contrast, HC abolished NO-induced relaxations while it did not affect those induced by GSNO, NH2OH and nerve activation. The catalase inhibitor 1,2,4 aminotriazole failed to affect nerve and NH2OH induced relaxations. These findings indicate that among the products that can be formed during NOS catalyzed L-arginine N(G)-oxidation, only NH2OH caused relaxations that exhibited a pharmacological profile similar to those induced by the nitrergic neurotransmitter. Furthermore, if NH2OH is the actual neurotransmitter it appears to be acting either directly or by a catalase independent release of NO.

Inhibitory effect of GnRH on isolated rat uterine muscle contractility.

The effect of GnRH upon uterine contractions of both non-pregnant and pregnant rats was examined in vitro. In the non-pregnant rat uterus, GnRH inhibited in a concentration-and-time dependent manner the contractions induced by acetylcholine and oxytocin, but not those caused by bradykinin and angiotensin II. GnRH also inhibited the rhythmic contractions induced by oxytocin in uterine strips from late pregnant rats. These findings show that GnRH has a direct inhibitory effect on the rat uterine contractions, suggesting that GnRH-like substances may exert modulatory influences upon rat uterine contractility.

Mechanism of pain induced by radiocontrast media.

In lightly-anesthetized dogs, ionic or non-ionic RCM (Iotalamato and iohexol, respectively) when injected by intracarotid route (i.c.), elicit a pain response comparable to that caused by bradykinin (BK) or capsaicin (CAP). This response, which is characterized by vocalization, hyperpnea, bradycardia and neck muscle contraction, was dose dependent and related to the osmolarity of the RCM. In the present study we observed that indomethacin did not interfere with CAP and RCM-induced pain at dose (2 mg/kg i.c.) that reduced BK-elicited responses. In contrast, Ruthenium Red (RR), in dose (1 mg/kg i.c.) that reduced CAP and/or RCM-induced effects did not affect BK-induced phenomena. We also verified that L-NAME (50 mg/kg i.c.) reduced the BK-, but not the CAP- and/or RCM-induced pain responses which suggests that an L-arginine-derived NO or related compound is involved in BK activation of perivascular nociceptors. Indeed, we found that i.c. injection of 20 mg of S-nitrosocysteine, a putative EDRF, caused BK-like responses. On the other hand, RCM and CAP appear to activate the same RR sensitive ionic channels of primary afferent endings. Therefore, RR-analogues could constitute a novel approach to minimizing or eventually abolishing the RCM side effects.