Pediatric pulmonary arterial hypertension due to a novel homozygous GDF2 missense variant affecting BMP9 processing and activity.

Pulmonary arterial hypertension (PAH) is a rare and severe disorder characterized by progressive pulmonary vasculopathy. Growth differentiation factor (GDF)2 encodes the pro-protein bone morphogenetic protein (BMP) 9, activated after cleavage by endoproteases into an active mature form. BMP9, together with BMP10, are high-affinity ligands of activin receptor-like kinase 1 (ALK1) and BMP receptor type II (BMPR2). GDF2 mutations have been reported in idiopathic PAH with most patients being heterozygous carriers although rare homozygous cases have been described. The link between PAH occurrence and BMP9 or 10 expression level is still unclear. In this study, we describe a pediatric case of PAH also presenting with telangiectasias and epistaxis. The patient carries the novel homozygous GDF2 c.946A > G mutation, replacing the first arginine of BMP9's cleavage site (R316) by a glycine. We show that this mutation leads to an absence of circulating mature BMP9 and mature BMP9-10 heterodimers in the patient's plasma although pro-BMP9 is still detected at a similar level as controls. In vitro functional studies further demonstrated that the mutation R316G hampers the correct processing of BMP9, leading to the secretion of inactive pro-BMP9. The heterozygous carriers of the variant were asymptomatic, similarly to previous reports, reinforcing the hypothesis of modifiers preventing/driving PAH development in heterozygous carriers.

The importance of supplementary immunisation activities to prevent measles outbreaks during the COVID-19 pandemic in Kenya.

BACKGROUND: The COVID-19 pandemic has disrupted routine measles immunisation and supplementary immunisation activities (SIAs) in most countries including Kenya. We assessed the risk of measles outbreaks during the pandemic in Kenya as a case study for the African Region. METHODS: Combining measles serological data, local contact patterns, and vaccination coverage into a cohort model, we predicted the age-adjusted population immunity in Kenya and estimated the probability of outbreaks when contact-reducing COVID-19 interventions are lifted. We considered various scenarios for reduced measles vaccination coverage from April 2020. RESULTS: In February 2020, when a scheduled SIA was postponed, population immunity was close to the herd immunity threshold and the probability of a large outbreak was 34% (8-54). As the COVID-19 contact restrictions are nearly fully eased, from December 2020, the probability of a large measles outbreak will increase to 38% (19-54), 46% (30-59), and 54% (43-64) assuming a 15%, 50%, and 100% reduction in measles vaccination coverage. By December 2021, this risk increases further to 43% (25-56), 54% (43-63), and 67% (59-72) for the same coverage scenarios respectively. However, the increased risk of a measles outbreak following the lifting of all restrictions can be overcome by conducting a SIA with ≥ 95% coverage in under-fives. CONCLUSION: While contact restrictions sufficient for SAR-CoV-2 control temporarily reduce measles transmissibility and the risk of an outbreak from a measles immunity gap, this risk rises rapidly once these restrictions are lifted. Implementing delayed SIAs will be critical for prevention of measles outbreaks given the roll-back of contact restrictions in Kenya.

Antibody responses to Bordetella pertussis and other childhood vaccines in infants born to mothers who received pertussis vaccine in pregnancy - a prospective, observational cohort study from the United Kingdom.

The maternal Tdap (tetanus, diphtheria and acellular pertussis) vaccination programme in the United Kingdom has successfully reduced cases of pertussis in young infants. In addition to prevention of pertussis cases, it is also important to investigate the persistence of maternal antibodies during infancy and the possible interference of maternal antibodies with infant responses to vaccines. We recruited mother-infant pairs from vaccinated and unvaccinated pregnancies and measured concentrations of immunoglobulin (Ig)G against pertussis toxin (PTx), filamentous haemagglutinin (FHA), pertactin (Prn), diphtheria toxin (DTx), tetanus toxoid (TTx) Haemophilus influenzae type b (Hib) and Streptococcus pneumoniae in mothers and infants at birth, and in infants at 7 weeks and at 5 months. Thirty-one mother-infant pairs were tested. Tdap-vaccinated women had significantly higher antibody against Tdap antigens, compared to unvaccinated women (DTx, P = 0·01; PTx, FHA, Prn and TTx, P < 0·001). All antibodies were actively transferred to the infants (transfer ratio  > 1) with higher transfer of DTx (P = 0·04) and TTx (P = 0·02) antibody in Tdap-vaccinated pregnancies compared to unvaccinated pregnancies. Infants from Tdap-vaccinated pregnancies had significantly elevated antibodies to all antigens at birth (P < 0.001) and at 7 weeks (FHA, Prn, TTx, P < 0·001; DTx, P = 0.01; PTx, P = 0·004) compared to infants from unvaccinated pregnancies. Infants from Tdap-vaccinated and -unvaccinated pregnancies had comparable antibody concentrations following primary pertussis immunization (PTx, P = 0·77; FHA, P = 0·58; Prn, P = 0·60; DTx, P = 0·09; TTx, P = 0·88). These results support maternal immunization as a method of protecting vulnerable infants during their first weeks of life.

Configurations of the Re-scan Confocal Microscope (RCM) for biomedical applications.

The new high-sensitive and high-resolution technique, Re-scan Confocal Microscopy (RCM), is based on a standard confocal microscope extended with a re-scan detection unit. The re-scan unit includes a pair of re-scanning mirrors that project the emission light onto a camera in a scanning manner. The signal-to-noise ratio of Re-scan Confocal Microscopy is improved by a factor of 4 compared to standard confocal microscopy and the lateral resolution of Re-scan Confocal Microscopy is 170 nm (compared to 240 nm for diffraction limited resolution, 488 nm excitation, 1.49 NA). Apart from improved sensitivity and resolution, the optical setup of Re-scan Confocal Microscopy is flexible in its configuration in terms of control of the mirrors, lasers and filters. Because of this flexibility, the Re-scan Confocal Microscopy can be configured to address specific biological applications. In this paper, we explore a number of possible configurations of Re-scan Confocal Microscopy for specific biomedical applications such as multicolour, FRET, ratio-metric (e.g. pH and intracellular Ca2+ measurements) and FRAP imaging.

High risk of a large measles outbreak despite 30 years of measles vaccination in The Netherlands.

Our aim was to assess progress towards measles elimination from The Netherlands by studying humoral measles immunity in the Dutch population. A population-based seroepidemiological study was conducted in 2006-2007 (N = 7900). Serum samples were analysed by a bead-based multiplex immunoassay. IgG levels ⩾0·2 IU/ml were considered protective. The overall seroprevalence in the Dutch population was 96%. However, 51% of socio-geographically clustered orthodox Protestant individuals aged <10 years were susceptible. Infants might be susceptible to measles between ages 4 months and 14 months, the age at which maternal antibodies have disappeared and the first measles, mumps, rubella (MMR) vaccination is administered, respectively. Waning of antibody concentrations was slower after the second MMR vaccination than after the first. The Netherlands is at an imminent risk of a measles outbreak in the orthodox Protestant minority. To prevent subsequent transmission to the general population, efforts to protect susceptible age groups are needed.

Improving Quality of Life with Brachytherapy for Urological Malignancies.

Brachytherapy for localised prostate, muscle-invasive bladder and penile cancer is well established, providing high tumour dose delivery and minimising normal tissue doses compared with external beam techniques. In prostate cancer, the main impact on quality of life relates to diminished sexual function and irritative or obstructive urinary symptoms, which are seen up to 15 years after treatment. Significant changes in bowel function are rare. Compared with radical prostatectomy or external beam radiotherapy, irritative or obstructive urinary symptoms are more prominent, whereas incontinence is less than after radical prostatectomy and bowel changes are less than after external beam radiotherapy. For muscle-invasive bladder cancer, when compared with radical cystectomy, although no difference is seen for urinary symptoms or fatigue, role and social functioning scores are higher and there is better post-treatment sexual function in both men and women. Compared with surgical treatment for penile cancer, brachytherapy results in better erectile function scores than after glansectomy and partial penectomy and high quality of life scores, with good satisfaction ratings for cosmetic appearance.

Relationship between serum gamma-glutamyltransferase and chronic kidney disease in the United States adult population. Findings from the National Health and Nutrition Examination Survey 2001-2006.

BACKGROUND AND AIMS: Elevated serum levels of gamma-glutamyltransferase (GGT) are a marker of liver injury, but may also be associated with other diseases and death. Currently, the association of serum GGT concentrations with chronic kidney disease has not been established in the U.S. general population. METHODS AND RESULTS: We performed a cross-sectional analysis of data from the National Health and Nutrition Examination Survey 2001 through 2006 and examined the association between serum GGT concentrations and chronic kidney disease in a nationally representative sample of 13,188 adults aged 20 years or older. Glomerular filtration rate (eGFR) was estimated using the Modification of Diet in Renal Disease formula. The prevalence of chronic kidney disease defined as eGFR <60 ml/min/1.73 m(2) or abnormal albuminuria in those with eGFR ≥60 ml/min/1.73 m(2) was 13.9% (n = 1842). Serum GGT elevation was associated with an increased odds of chronic kidney disease (odds ratio 2.38, 95% confidence intervals 2.02-2.80, p<0.0001). After adjustment for demographics, comorbidities, daily alcohol consumption, lipid-lowering medications, viral hepatitis status and laboratory measures, the odds ratio of chronic kidney disease per log serum GGT increase was 1.79 (1.41, 2.27; p<0.0001). CONCLUSIONS: These results show a strong, independent, relationship of increased serum GGT concentrations with chronic kidney disease in the US adult population.

A ß-Carbon elimination strategy for convenient in situ access to cyclopentadienyl metal complexes.

The electronic and steric properties of tailored cyclopentadienyl (Cp) ligands are powerful handles to modulate the catalytic properties of their metal complexes. This requires the individual preparation, purification and storage of each ligand/metal combination. Alternative, ideally in situ, complexation protocols would be of high utility. We disclose a new approach to access Cp metal complexes. Common metal precursors rapidly react with cyclopentadienyl carbinols via ß-carbon eliminations to directly give the Cp-metal complexes. An advantage of this is the direct and flexible use of storable pre-ligands. No auxiliary base is required and the Cp complexes can be prepared in situ in the reaction vessel for subsequent catalytic transformations.

Presence and absence of follicle-stimulating hormone receptor mutations provide some insights into spontaneous ovarian hyperstimulation syndrome physiopathology.

CONTEXT: Ovarian hyperstimulation syndrome (OHSS) is a potentially life-threatening complication of ovarian stimulation treatments. Moreover, four mutations of the FSH receptor (FSHr) were recently described in patients presenting with spontaneous OHSS (sOHSS) of the first trimester of pregnancy with normal levels of human chorionic gonadotropin (hCG). OBJECTIVE: The objective of this study was to look for novel FSHr mutations in patients with sOHSS associated with different levels of hCG and TSH to 1) find new residues important for FSHr activation and specificity, and 2) better delineate the pathophysiology of the different presentations of sOHSS. DESIGN, INTERVENTION, AND PATIENTS: After blood sampling, we sequenced the FSHr from genomic leukocytes DNA from eight patients with sOHSS of the first or second trimester of pregnancy with normal or high hCG levels or with high TSH levels associated with severe hypothyroidism. SETTING: This study was performed at a university laboratory. MAIN OUTCOME MEASURE: The main outcome measure was FSHr sequencing and in vitro evaluation of the variation of cAMP production of FSHr mutants. RESULTS: A new mutation was found in the patient with sOHSS of the first trimester of pregnancy with a normal hCG level: I5.54(545)T, in transmembrane helix V of the FSHr. When tested functionally, this mutant displayed promiscuous activation by both hCG and TSH together with detectable constitutive activity. In contrast, no mutations were found in the FSHr from patients with high hCG or TSH levels, indicating that for those seven patients, sOHSS results from the natural promiscuous stimulation of a wild-type FSHr by very high concentrations of hCG or TSH. CONCLUSIONS: sOHSS can occur by at least three different pathophysiological mechanisms.

Cell wall dynamics in yeast.

The yeast Saccharomyces cerevisiae is the first fungus for which the structure of the cell wall is known at the molecular level. It is a dynamic and highly regulated structure. This is vividly illustrated when the cell wall is damaged and a salvage pathway becomes active, resulting in compensatory changes in the wall.

Reversal of low dose angiotension hypertension by angiotensin receptor antagonists.

During acute angiotension II (Ang II) infusion (200 ng/kg/min i.v.) into anesthetized rats, mean arterial pressure rose from 124 +/- 1 to 154 +/- 2 mm Hg. The peptidic Ang II antagonist saralasin lowered arterial pressure in a dose-dependent manner. The maximal decrease in pressure was similar to that observed after the Ang II infusion was discontinued. The nonpeptide Ang II antagonist, 4'-[( 2-butyl-4-chloro-5-(hydroxymethyl)-1H-imidazole-1-yl] methyl) [1,1'-biphenyl] -2-carboxylic acid (SC-48742), lowered acutely elevated arterial pressure to a level similar to that on discontinuation of the angiotensin infusion. Chronic (8 days) infusion of Ang II (20 ng/kg/min i.v.) increased mean arterial pressure from 116 +/- 3 to 164 +/- 7 mm Hg, which then decreased to 121 +/- 6 mm Hg on termination of the infusion. Saralasin (10 micrograms/kg/min, a maximally effective dose during acute angiotensin infusion) decreased mean arterial pressure from 168 +/- 7 to 141 +/- 3 mm Hg, a pressure significantly higher (p less than 0.05) than the pressure observed after the angiotensin infusion was discontinued. SC-48742 decreased mean arterial pressure from 167 +/- 7 to 127 +/- 3 mm Hg, a pressure not statistically different from the minimum pressure observed after the angiotensin infusion was terminated. The mechanism of blood pressure elevation during acute high dose or chronic low dose Ang II infusion is different, the latter having a significant neural component as measured by the response to trimethaphan. The peptidic antagonist saralasin was fully effective in lowering acute angiotensin hypertension but only partially effective during chronic hypertension.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin subtype 1 blockade selectively potentiates adenosine subtype 2-mediated vasodilation.

A previous report demonstrated that infusion of adenosine into the forearm increased local vascular production of angiotensin II. We hypothesize that this increase in angiotensin II could attenuate the vasodilator response to adenosine subtype 2 (A2) receptor activation. The depressor and regional hemodynamic responses to the A2-selective adenosine agonist DPMA were measured in the presence and absence of angiotensin subtype 1 (AT1) receptor blockade (losartan, 10 mg/kg IV) in anesthetized rats. Losartan pretreatment (without versus with losartan) significantly potentiated DPMA-induced reductions in renal (-13 +/- 2% versus -22 +/- 4%, P < .05) and mesenteric (-11 +/- 2% versus -23 +/- 4%, P < .05) vascular resistances, resulting in a greater depressor response (-7 +/- 2 versus -18 +/- 3 mm Hg, P < .05). The decrease in hindquarter vascular resistance was not affected. To test the specificity of this interaction, we also evaluated nitroglycerin and nifedipine. Pretreatment with losartan had no effect on the responses to nitroglycerin, whereas the responses to nifedipine either were not affected or were attenuated (percent change in mesenteric vascular resistance: without losartan pretreatment, -30 +/- 1%; with losartan pretreatment, -24 +/- 2%, P < .05). To determine whether the decrease in arterial pressure after losartan pretreatment contributed to the potentiation of the DPMA-mediated effects, we infused nitroglycerin to lower mean arterial pressure comparably to losartan treatment. None of the hemodynamic responses to subsequent DPMA administration were affected. These data suggest that endogenous levels of angiotensin II, whether released locally or systemically, selectively attenuate the A2-mediated reductions in renal and mesenteric vascular resistances.

Synthesis and structure-activity relationships of nonpeptide, potent triazolone-based angiotensin II receptor antagonists.

2,5-Dibutyl-2,4-dihydro-4-[[2-(1H-tetrazol-5-yl)[1,1'-biphenyl]-4' - yl]methyl]-3H-1,2,4-triazol-3-one, SC-51316, was synthesized as a potent and orally active angiotensin II (AII) receptor antagonist with a long duration of action. To explore the lipophilic pocket in the AII receptor interacting with the substituent at the 2-position of triazolone-based antagonists, a series of compounds were prepared and evaluated for receptor binding affinity and antagonism of AII-contracted rabbit aortic rings. It has been found that the pocket is very spacious and can accommodate different sizes of lipophilic groups and various functionalities. Acidic groups generally result in a slight decrease in binding affinity. Branched chains are unfavorable. The freedom of rotation around C2-C3 in the flexible side chain is crucial for good binding. The 2-phenylethyl-substituted triazolone analogue exhibits the highest in vitro potency among all compounds that have been synthesized.

Structure-activity relationships for the carboxy-terminus truncated analogues of angiotension II, a new class of angiotensin II antagonists.

A series of analogues of the recently reported angiotensin II (AII) antagonist [Sar1]AII-(1-7)-amide or des-Phe8[Sar1]AII (3) have been prepared by solid-phase synthesis and purified by reverse-phase liquid chromatography. The agonist and antagonist properties of these carboxy-truncated analogues of AII were determined in the isolated rabbit aorta assay. In the analogues tested, replacement of aspartic acid in position 1 by sarcosine was found necessary to produce significant antagonist activity. At position 7 of the des-Phe8 analogues, prolinamide could be replaced by proline without significant change in the biological activity. However, substitution of 7-prolinamide by either glycinamide or sarcosinamide provided inactive peptides. Methylation of the 4-tyrosine in [Sar1]AII-(1-7)-NH2 preserved the antagonist potency in isolated rabbit aorta. Deletion of the proline at position 7 resulted in inactive hexapeptides, both in the Asp1 and Sar1 series. However synthesis of the N,N-dimethyl amide at the N-terminus afforded hexapeptide [Sar1]AII-(1-6)-N(CH3)2 (10) with a pA2 value of 7.05. All the antagonistic peptides synthesized were fully reversible, competitive antagonists in vitro. These findings indicate that the structural requirements for receptor blockade by these C-truncated analogues are quite stringent with respect to the nature of the amino acid at positions 1 and 6/7. The analogues 2, 3, 7, 10, 11 (saralasin), and 12 (sarmesin) were tested in vivo in the anesthetized rat and were found to inhibit the AII pressor response. In addition, 3 inhibited angiotensin II stimulated aldosterone release from isolated rat adrenal zona glomerulosa cells and had no agonist activity by itself at the doses tested. Interestingly, analogue 3, when injected intracerebroventricularly in conscious rats, failed to antagonize the dipsogenic response to an angiotensin II icv injection and this reflects some heterogeneity in the AII receptor population. Peptide 3 is the first example of an antagonist that discriminates between peripheral and brain receptor subtypes.

Nonpeptide angiotensin II antagonists: N-phenyl-1H-pyrrole derivatives are angiotensin II receptor antagonists.

A series of 5-[1-[4-[(4,5-disubstituted-1H-imidazol-1-yl)methyl]- substituted]-1H-pyrrol-2-yl]-1H-tetrazoles and 5-[1-[4-[(3,5-dibutyl-1H-1,2,4-triazol-1-yl)methyl]-substituted]- 1H-pyrrol-2-yl]-1H-tetrazoles were investigated as novel AT1-selective angiotensin II receptor antagonists. Computer-assisted modeling techniques were used to evaluate structural parameters in comparison to the related biphenyl system. New synthetic procedures have been developed to prepare the novel compounds. The best antagonists in this series had IC50 values (rat uterine membrane receptor binding) in the 10(-8) M range and corresponding pA2 in isolated organ assay (rabbit aorta rings). Structure-activity relationships indicate some similarities with the finding in the biphenyl system. Substitution on the pyrrole ring modulates activity. Compound 5 antagonized angiotensin-induced blood pressure increase when administered to conscious rat at 30 mg/kg per os.

Influence of age on the signal transduction pathway of non-adrenergic non-cholinergic neurotransmitters in the rat gastric fundus.

1. The influence of aging on the relaxant response and the change in cyclic nucleotide content induced by vasoactive intestinal polypeptide (VIP), nitric oxide (NO), electrical field stimulation of the non-adrenergic non-cholinergic neurones and substances acting at different levels of the cyclic AMP and cyclic GMP transduction pathways was studied in longitudinal muscle strips of the rat gastric fundus. 2. The relaxant responses to VIP, sustained electrical stimulation, forskolin and 3-isobutyl-1-methylxanthine were reduced with age, while the responses to dibutyryl cyclic AMP were not. The increase in cyclic AMP content induced by sustained electrical stimulation and forskolin was lower in old rats. 3. The relaxant responses to NO and to short train electrical stimulation were similar in the three age groups. The inhibitory effect of NG-nitro-L-arginine methyl ester on relaxations induced by short train electrical stimulation was more pronounced in old rats. The relaxant responses to sodium nitroprusside (SNP), 8-bromo-cyclic GMP and zaprinast were reduced with age. SNP induced a similar elevation of the cyclic GMP content in the three age groups. 4. These results suggest that aging differentially affects the cyclic AMP and cyclic GMP pathway for relaxation by VIP and NO in the rat gastric fundus, as the defect seems to occur at the level of the adenylate cyclase and cyclic GMP-dependent protein kinase respectively.

Modulation of non-adrenergic non-cholinergic inhibitory neurotransmission in rat gastric fundus by the alpha 2-adrenoceptor agonist, UK-14,304.

1. The influence of the alpha 2-adrenoceptor agonist, UK-14,304, on non-adrenergic non-cholinergic (NANC) relaxation induced by electrical field stimulation was investigated in longitudinal muscle strips of the gastric fundus of reserpinized rats. 2. In tissues where tone was raised by 3 x 10(-7) M prostaglandin F2 alpha (PGF2 alpha), the inhibitory effect of 10(-6) M UK-14,304, on the NANC relaxations induced by short train stimulation (40 V, 1 ms, 20 s) was inversely related to the stimulus frequency (1-4-16 Hz). UK-14,304 (10(-6) M) did not influence relaxations induced by administration of exogenous nitric oxide (NO, 2 x 10(-6) M-10(-4) M). The inhibitory effect of UK-14,304 on the electrically induced relaxations was antagonized by 10(-6) M rauwolscine but not by 10(-6) M prazosin. 3. UK-14,304 (10(-6) M) also reduced the amplitude of the sustained NANC relaxation, induced by electrical field stimulation (40 V, 1 ms, 4 Hz) for 5 min. The effect of UK-14,304 was also antagonized by 10(-6) M rauwolscine but not by 10(-6) M prazosin. UK-14,304 (10(-6) M) did not reduce the relaxation induced by 3 x 10(-9) M vasoactive intestinal polypeptide (VIP). 4. These results suggest that the release of the inhibitory NANC neurotransmitter during short train stimulation, thought to be NO, and during sustained stimulation, thought to be VIP, is inhibited by stimulation of presynaptic alpha 2-adrenoceptors in the rat gastric fundus.

Development of cholinergic and inhibitory non-adrenergic non-cholinergic responses in the rat gastric funds.

1. Cholinergic contractions and inhibitory non-adrenergic non-cholinergic (NANC) relaxations were studied in longitudinal muscle strips of the gastric funds of 2, 4 and 8 week old rats. 2. Contractions induced by electrical stimulation of the cholinergic neurones and by administration of acetylcholine decreased during development. The potentiating effect of physostigmine was similar in the 3 age groups. 3. Short train stimulation in NANC conditions induced fast relaxations, which were more pronounced in 4 and 8 week than in 2 week old rats. These relaxations were almost completely inhibited by NG-nitro-L-arginine methyl ester (L-NAME) in the 3 age groups. The nitric oxide-induced relaxations did not change during development. 4. Sustained electrical stimulation in NANC conditions induced an initial relaxation, which was almost totally blocked by L-NAME, followed by an almost complete recovery of tone at lower frequencies of stimulation. At higher frequencies of stimulation, the recovery of tone was incomplete or absent. This sustained relaxation was only partially reduced by L-NAME and almost abolished by L-NAME plus alpha-chymotrypsin. The initial relaxations increased during development, while the sustained relaxations remained similar during this period. Vasoactive intestinal polypeptide-induced relaxations were also similar in the 3 age groups. 5. These results show that the sensitivity of the gastric fundus to acetylcholine decreases from 2 weeks to 8 weeks postnatally, while the importance of the nitrergic innervation increases during this period.

ATP and nitric oxide: inhibitory NANC neurotransmitters in the longitudinal muscle-myenteric plexus preparation of the rat ileum.

1. The nature of neurotransmitter(s) involved in non-adrenergic non-cholinergic (NANC) relaxations induced by electrical stimulation (10 s trains, 1-8 Hz) was investigated in the precontracted longitudinal muscle-myenteric plexus preparation of the rat ileum. 2. Electrical stimulation of the tissue induced complex responses, consisting of a primary contraction, a primary relaxation, an off-relaxation and a rebound contraction, which were all tetrodotoxin(TTX)-sensitive. 3. Vasoactive intestinal polypeptide (VIP) and carbon monoxide (CO) did not induce relaxations. alpha-Chymotrypsin did not reduce the relaxations induced by electrical stimulation, while zinc protoporphyrin IX had non-specific effects. 4. Nitric oxide (NO) induced concentration-dependent relaxations. NG-nitro-L-arginine methylester (L-NAME) abolished the primary contractions and off-relaxations, while it partially reduced the primary relaxations. 5. ATP induced relaxations and ATP-desensitization of the tissues partially reduced the primary relaxations. Suramin and reactive blue 2 did not consistently influence the primary relaxations. 6. The ATP-induced relaxations were not influenced by L-NAME or TTX. The inhibitory effect of ATP-desensitization and L-NAME did not summate. 7. The cyclic AMP content of the tissue did not increase upon electrical stimulation or after addition of NO or ATP. The cyclic GMP content of the tissue increased upon electrical stimulation and addition of NO, but not after addition of ATP. 8. It is concluded that the relaxation induced by electrical stimulation consists of two types of responses. The off-relaxation is completely nitrergic, while the primary relaxation is mediated by NO, ATP and an as yet unknown transmitter which is not VIP or CO.