Possible involvement of A1 receptors in the inhibition of gonadotropin secretion induced by adenosine in rat hemipituitaries in vitro

We investigated the participation of A1 or A2 receptors in the gonadotrope and their role in the regulation of LH and FSH secretion in adult rat hemipituitary preparations, using adenosine analogues. A dose-dependent inhibition of LH and FSH secretion was observed after the administration of graded doses of the R-isomer of phenylisopropyladenosine (R-PIA; 1 nM, 10 nM, 100 nM, 1 µM and 10 µM). The effect of R-PIA (10 nM) was blocked by the addition of 8-cyclopentyltheophylline (CPT), a selective A1 adenosine receptor antagonist, at the dose of 1 µM. The addition of an A2 receptor-specific agonist, 5-N-methylcarboxamidoadenosine (MECA), at the doses of 1 nM to 1 µM had no significant effect on LH or FSH secretion, suggesting the absence of this receptor subtype in the gonadotrope. However, a sharp inhibition of the basal secretion of these gonadotropins was observed after the administration of 10 µM MECA. This effect mimicked the inhibition induced by R-PIA, supporting the hypothesis of the presence of A1 receptors in the gonadotrope. R-PIA (1 nM to 1 µM) also inhibited the secretion of LH and FSH induced by phospholipase C (0.5 IU/ml) in a dose-dependent manner. These results suggest the presence of A1 receptors and the absence of A2 receptors in the gonadotrope. It is possible that the inhibition of LH and FSH secretion resulting from the activation of A1 receptors may have occurred independently of the increase in membrane phosphoinositide synthesis.

Stimulatory effects of adenosine on prolactin secretion in the pituitary gland of the rat

We investigated the effects of adenosine on prolactin (PRL) secretion from rat anterior pituitaries incubated in vitro. The administration of 5-N-methylcarboxamidoadenosine (MECA), an analog agonist that preferentially activates A2 receptors, induced a dose-dependent (1 nM to 1 µM) increase in the levels of PRL released, an effect abolished by 1,3-dipropyl-7-methylxanthine, an antagonist of A2 adenosine receptors. In addition, the basal levels of PRL secretion were decreased by the blockade of cyclooxygenase or lipoxygenase pathways, with indomethacin and nordihydroguaiaretic acid (NDGA), respectively. The stimulatory effects of MECA on PRL secretion persisted even after the addition of indomethacin, but not of NDGA, to the medium. MECA was unable to stimulate PRL secretion in the presence of dopamine, the strongest inhibitor of PRL release that works by inducing a decrease in adenylyl cyclase activity. Furthermore, the addition of adenosine (10 nM) mimicked the effects of MECA on PRL secretion, an effect that persisted regardless of the presence of LiCl (5 mM). The basal secretion of PRL was significatively reduced by LiCl, and restored by the concomitant addition of both LiCl and myo-inositol. These results indicate that PRL secretion is under a multifactorial regulatory mechanism, with the participation of different enzymes, including adenylyl cyclase, inositol-1-phosphatase, cyclooxygenase, and lipoxygenase. However, the increase in PRL secretion observed in the lactotroph in response to A2 adenosine receptor activation probably was mediated by mechanisms involving regulation of adenylyl cyclase, independent of membrane phosphoinositide synthesis or cyclooxygenase activity and partially dependent on lipoxygenase arachidonic acid-derived substances.

The effects of 2,3-diphosphoglycerate, adenosine triphosphate, and glycosylated hemoglobin on the hemoglobin-oxygen affinity of diabetic patients

The position of the oxygen dissociation curve (ODC) is modulated by 2,3-diphosphoglycerate (2,3-DPG). Decreases in 2,3-DPG concentration within the red cell shift the curve to the left, whereas increases in concentration cause a shift to the right of the ODC. Some earlier studies on diabetic patients have reported that insulin treatment may reduce the red cell concentrations of 2,3-DPG, causing a shift of the ODC to the left, but the reports are contradictory. Three groups were compared in the present study: 1) nondiabetic control individuals (N = 19); 2) insulin-dependent diabetes mellitus (IDDM) patients (on insulin treatment) (N = 19); 3) non-insulin-dependent diabetes mellitus (NIDDM) patients using oral hypoglycemic agents and no insulin treatment (N = 22). The overall position of the ODC was the same for the three groups despite an increase of the glycosylated hemoglobin fraction that was expected to shift the ODC to the left in both groups of diabetic patients (HbA1c: control, 4.6%; IDDM, 10.5%; NIDDM, 9.0%). In IDDM patients, the effect of the glycosylated hemoglobin fraction on the position of the ODC appeared to be counterbalanced by small though statistically significant increases in 2,3-DPG concentration from 2.05 (control) to 2.45 µmol/ml blood (IDDM). Though not statistically significant, an increase of 2,3-DPG also occurred in NIDDM patients, while red cell ATP levels were the same for all groups. The positions of the ODC were the same for control subjects, IDDM and NIDDM patients. Thus, the PO2 at 50% hemoglobin-oxygen saturation was 26.8, 28.2 and 28.5 mmHg for control, IDDM and NIDDM, respectively. In conclusion, our data question the idea of adverse side effects of insulin treatment on oxygen transport. In other words, the shift to the left reported by others to be caused by insulin treatment was not detected.

Adenosine A1 receptor-mediated inhibition of in vitro prolactin secretion from the rat anterior pituitary

In previous studies, we demonstrated biphasic purinergic effects on prolactin (PRL) secretion stimulated by an adenosine A2 agonist. In the present study, we investigated the role of the activation of adenosine A1 receptors by (R)-N6-(2-phenylisopropyl)adenosine (R-PIA) at the pituitary level in in vitro PRL secretion. Hemipituitaries (one per cuvette in five replicates) from adult male rats were incubated. Administration of R-PIA (0.001, 0.01, 0.1, 1, and 10 µM) induced a reduction of PRL secretion into the medium in a U-shaped dose-response curve. The maximal reduction was obtained with 0.1 µM R-PIA (mean ± SEM, 36.01 ± 5.53 ng/mg tissue weight (t.w.)) treatment compared to control (264.56 ± 15.46 ng/mg t.w.). R-PIA inhibition (0.01 µM = 141.97 ± 15.79 vs control = 244.77 ± 13.79 ng/mg t.w.) of PRL release was blocked by 1 µM cyclopentyltheophylline, a specific A1 receptor antagonist (1 µM = 212.360 ± 26.560 ng/mg t.w.), whereas cyclopentyltheophylline alone (0.01, 0.1, 1 µM) had no effect. R-PIA (0.001, 0.01, 0.1, 1 µM) produced inhibition of PRL secretion stimulated by both phospholipase C (0.5 IU/mL; 977.44 ± 76.17 ng/mg t.w.) and dibutyryl cAMP (1 mM; 415.93 ± 37.66 ng/mg t.w.) with nadir established at the dose of 0.1 µM (225.55 ± 71.42 and 201.9 ± 19.08 ng/mg t.w., respectively). Similarly, R-PIA (0.01 µM) decreased (242.00 ± 24.00 ng/mg t.w.) the PRL secretion stimulated by cholera toxin (0.5 mg/mL; 1050.00 ± 70.00 ng/mg t.w.). In contrast, R-PIA had no effect (468.00 ± 34.00 ng/mg t.w.) on PRL secretion stimulation by pertussis toxin (0.5 mg/mL; 430.00 ± 26.00 ng/mg t.w.). These results suggest that inhibition of PRL secretion after A1 receptor activation by R-PIA is mediated by a Gi protein-dependent mechanism.

Antidipsogenic effects of central adenosine-5'-triphosphate

Besides other physiological functions, adenosine-5'-triphosphate (ATP) is also a neurotransmitter that acts on purinergic receptors. In spite of the presence of purinergic receptors in forebrain areas involved with fluid-electrolyte balance, the effect of ATP on water intake has not been investigated. Therefore, we studied the effects of intracerebroventricular (icv) injections of ATP (100, 200 and 300 nmol/µL) alone or combined with DPCPX or PPADS (P1 and P2 purinergic antagonists, respectively, 25 nmol/µL) on water intake induced by water deprivation. In addition, the effect of icv ATP was also tested on water intake induced by intragastric load of 12% NaCl (2 mL/rat), acute treatment with the diuretic/natriuretic furosemide (20 mg/kg), icv angiotensin II (50 ng/µL) or icv carbachol (a cholinergic agonist, 4 nmol/µL), on sodium depletion-induced 1.8% NaCl intake, and on food intake induced by food deprivation. Male Holtzman rats (280-320 g, N = 7-11) had cannulas implanted into the lateral ventricle. Icv ATP (300 nmol/µL) reduced water intake induced by water deprivation (13.1 ± 1.9 vs saline: 19.0 ± 1.4 mL/2 h; P < 0.05), an effect blocked by pre-treatment with PPADS, but not DPCPX. Icv ATP also reduced water intake induced by NaCl intragastric load (5.6 ± 0.9 vs saline: 10.3 ± 1.4 mL/2 h; P < 0.05), acute furosemide treatment (0.5 ± 0.2 vs saline: 2.3 ± 0.6 mL/15 min; P < 0.05), and icv angiotensin II (2.2 ± 0.8 vs saline: 10.4 ± 2.0 mL/2 h; P < 0.05), without changing icv carbachol-induced water intake, sodium depletion-induced 1.8% NaCl intake and food deprivation-induced food intake. These data suggest that central ATP, acting on purinergic P2 receptors, reduces water intake induced by intracellular and extracellular dehydration.

Molecular characterization of environmental Cryptococcus neoformans isolated in Vitoria, ES, Brazil

Cryptococcus neoformans is the major cause of fungal meningitis, a potentially lethal mycosis. Bird excreta can be considered a significant environmental reservoir of this species in urban areas, thirty-three samples of pigeon excreta were collected within the city of Vitoria, Brazil. Cryptococcus neoformans was isolated and identified using standard biochemical assays in ten samples. PCR amplification with primer M13 and orotidine monophosphate pyrophosphorylase (URA5) gene-restriction fragment length polymorphism (RFLP) analysis discerned serotypes and genotypes within this species. All isolates were serotype A (C. neoformans var. grubii) and genotype VNI. The two alternative alleles a and α at the mating type locus were determined by PCR amplification and mating assays performed on V8 medium. All isolates were MAT α mating type but only 50% were able to mate in vitro with the opposite mating type MAT a tester strains (JEC20, KN99a and Bt63). This study adds information on the ecology and molecular characterization of C. neoformans in the Southeast region of Brazil.

Aminoquinolone WR6026 as a feasible substitute for gentian violet in Chagas' disease prophylaxis in preserved blood for transfusional purposes

The search for a colorless, nontoxic and efficient drug to prevent transfusion-associated Chagas' disease (TACD) has been underway unsuccessfully since 1953 when gentian violet was preconized and to date is still being used as the only in vitro trypanocidal agent. The recent findings of aminoquinolone "WR6026" as a trypanocidal agent, led the authors to study the metabolism of red cells stored with this compound, the main objective of which was to define its applicability in TACD control. Ten units of human whole blood collected in CPDA-1 were divided into two equal satellite bags. One had "WR6026" (final concentration 62.5µg/mL) added and the other was used as a control, both were stored at 4ºC. At baseline, day 7, 14, 21 and 28, samples were taken for the following measurements: adenosine triphosphate (ATP), hemoglobin, electrolytes (sodium and potassium), gases (pO2 and pCO2) and osmotic fragility. The results of tests and control were analyzed through parametric t-student test. The results were similar in both groups throughout the experiment except for the level of ATP on day 14, which presented significantly higher values in the tests when compared with the controls (p = 0.012). It was concluded that WR6026 does not interfere in the preservation and probably the viability of the erythrocytes also until day 28 of storage. Consequently the authors suggest that WR6026 could emerge as a colorless substitute for gentian violet in the control of TACD in endemic areas.

Influence of caffeine on blood pressure and platelet aggregation

OBJECTIVE: Studies have demonstrated that methylxanthines, such as caffeine, are A1 and A2 adenosine receptor antagonists found in the brain, heart, lungs, peripheral vessels, and platelets. Considering the high consumption of products with caffeine in their composition, in Brazil and throughout the rest of the world, the authors proposed to observe the effects of this substance on blood pressure and platelet aggregation. METHODS: Thirteen young adults, ranging from 21 to 27 years of age, participated in this study. Each individual took 750mg/day of caffeine (250mg tid), over a period of seven days. The effects on blood pressure were analyzed through the pressor test with handgrip, and platelet aggregation was analyzed using adenosine diphosphate, collagen, and adrenaline. RESULTS: Diastolic pressure showed a significant increase 24 hours after the first intake (p<0.05). This effect, however, disappeared in the subsequent days. The platelet aggregation tests did not reveal statistically significant alterations, at any time during the study. CONCLUSION: The data suggest that caffeine increases diastolic blood pressure at the beginning of caffeine intake. This hypertensive effect disappears with chronic use. The absence of alterations in platelet aggregation indicates the need for larger randomized studies.

Study of Coronary Sinus Flow Reserve Through Transesophageal Doppler Echocardiography in Normal Subjects

OBJECTIVE - To evaluate the Coronary Flow Reserve in the Coronary Sinus through transesophageal Doppler echocardiography in normal subjects. METHODS - We obtained technically adequate flow samples for analysis in 10 healthy volunteers (37±8 years, 5 men) with no history of heart or systemic disease and with mean left ventricular mass index by transthoracic echocardiography of 87±18 g/m². Coronary sinus flow velocity was recorded within the coronary sinus with the patient in a resting condition and during intravenous adenosine infusion at a dose of 140 µg/kg/min for 4 minutes. Recording of coronary sinus blood flow was possible in all cases with measurement of peak systolic, diastolic, and retrograde velocities (PSV, PDV, and PRV, cm/sec), mean systolic and diastolic velocities (MSV and MDV, cm/sec), and systolic and diastolic velocity time integral (VTI S and VTI D, cm/sec). RESULTS - The coronary flow reserve was calculated as the ratio between the blood flow in the basal state and the maximum measured hyperemic blood flow with adenosine infusion. Results are shown as mean and standard deviations. (CFR = PSV + PDV -- PRV/basal PSV): 1st min = 2.2±0.21; 2nd min = 3±0.3; 3rd min = 3.4±0.37; 4th min = 3.6 ± 0.33. CONCLUSION - Although coronary sinus flow had significantly increased in the first minute, higher velocities were seen at third and fourth minutes, indicating that these should be the best times to study coronary sinus flow with intravenous adenosine in continuous infusion.

Effect of selective phosphodiesterase inhibitors on the rat eosinophil chemotactic response in vitro

In the present study, we have performed a comparative analysis of the effect of selective inhibitors of phosphodiesterase (PDE) type III, IV and V on eosinophil chemotaxis triggered by platelet activating factor (PAF) and leukotriene B4 (LTB4) in vitro. The effect of the analogues N6-2'-O-dibutyryladenosine 3':5' cyclic monophosphate (Bt2 cyclic AMP) and N2-2'-O- dibutyrylguanosine 3':5' cyclic monophosphate (Bt2 cyclic GMP) has also been determined. The eosinophils were obtained from the peritoneal cavity of naive Wistar rats and purified in discontinuous Percoll gradients to 85-95% purity. We observed that pre-incubation of eosinophils with the PDE type IV inhibitor rolipram suppressed the chemotactic response triggered by PAF and LTB4, in association with an increase in the intracellular levels of cyclic AMP. In contrast, neither zaprinast (type V inhibitor) nor type III inhibitors milrinone and SK&F 94836 affected the eosinophil migration. Only at the highest concentration tested did the analogue Bt2 cyclic AMP suppress the eosinophil chemotaxis, under conditions where Bt2 cyclic GMP was ineffective. We have concluded that inhibition of PDE IV, but not PDE III or V, was able to block the eosinophil chemotaxis in vitro, suggesting that the suppressive activity of selective PDE IV inhibitors on tissue eosinophil accumulation may, at least, be partially dependent on their ability to directly inhibit the eosinophil migration.

P2X1 receptors and the endothelium

Adenosine triphosphate (ATP) is now established as a principle vaso-active mediator in the vasculature. Its actions on arteries are complex, and are mediated by the P2X and P2Y receptor families. It is generally accepted that ATP induces a bi-phasic response in arteries, inducing contraction via the P2X and P2Y receptors on the smooth muscle cells, and vasodilation via the actions of P2Y receptors located on the endothelium. However, a number of recent studies have placed P2X1 receptors on the endothelium of some arteries. The use of a specific P2X1 receptor ligand, a, b methylene ATP has demonstrated that P2X1 receptors also have a bi-functional role. The actions of ATP on P2X1 receptors is therefore dependant on its location, inducing contraction when located on the smooth muscle cells, and dilation when expressed on the endothelium, comparable to that of P2Y receptors.

Molecular and biochemical characterisation of Trypanosoma cruzi phosphofructokinase

The characterisation of the gene encoding Trypanosoma cruzi CL Brener phosphofructokinase (PFK) and the biochemical properties of the expressed enzyme are reported here. In contradiction with previous reports, the PFK genes of CL Brener and YBM strain T. cruzi were found to be similar to their Leishmania mexicana and Trypanosoma brucei homologs in terms of both kinetic properties and size, with open reading frames encoding polypeptides with a deduced molecular mass of 53,483. The predicted amino acid sequence contains the C-terminal glycosome-targeting tripeptide SKL; this localisation was confirmed by immunofluorescence assays. In sequence comparisons with the genes of other eukaryotes, it was found that, despite being an adenosine triphosphate-dependent enzyme, T. cruzi PFK shows significant sequence similarity with inorganic pyrophosphate-dependent PFKs.

Analysis of the NTPDase and ecto-5'-nucleotidase profiles in serum-limited Trichomonas vaginalis

Trichomonas vaginalis is a parasite of the human urogenital tract that causes trichomonosis, the most prevalent non-viral sexually transmitted disease. Ectonucleoside triphosphate diphosphohydrolase (NTPDase) family members, which hydrolyse extracellular ATP and ADP and ecto-5′-nucleotidase, which hydrolyses AMP, have been characterised in T. vaginalis. For trichomonad culture, the growth medium is supplemented with 10% serum, which is an important source of nutrients, such as adenosine. Here, we investigated the ATP metabolism of T. vaginalis trophozoites from long-term cultures and clinical isolates under limited bovine serum conditions (1% serum). The specific enzymatic activities were expressed as nmol inorganic phosphate (Pi) released/min/mg protein, the gene expression patterns were determined by reverse transcriptase-polymerase chain reaction, the extracellular adenine nucleotide hydrolysis was analysed by high performance liquid chromatography and the cell cycle analysis was assessed by flow cytometry. Serum limitation led to the profound activation of NTPDase and ecto-5'-nucleotidase activities. Furthermore, the levels of NTPDase A and B transcripts increased and extracellular ATP metabolism was activated, which led to enhanced ATP hydrolysis and the formation of ADP and AMP. Moreover, the cell cycle was arrested at the G0/G1 stage, which suggested adenosine uptake. Our data suggest that under conditions of serum limitation, NTPDase and ecto-5'-nucleotidase play a role in providing the adenosine required for T. vaginalis growth and that this process contributes to the establishment of parasitism.

The zinc finger protein TcZFP2 binds target mRNAs enriched during Trypanosoma cruzi metacyclogenesis

Trypanosomes are parasitic protozoa in which gene expression is primarily controlled through the regulation of mRNA stability and translation. This post-transcriptional control is mediated by various families of RNA-binding proteins, including those with zinc finger CCCH motifs. CCCH zinc finger proteins have been shown to be essential to differentiation events in trypanosomatid parasites. Here, we functionally characterise TcZFP2 as a predicted post-transcriptional regulator of differentiation in Trypanosoma cruzi. This protein was detected in cell culture-derived amastigotes and trypomastigotes, but it was present in smaller amounts in metacyclic trypomastigote forms of T. cruzi. We use an optimised recombinant RNA immunopreciptation followed by microarray analysis assay to identify TcZFP2 target mRNAs. We further demonstrate that TcZFP2 binds an A-rich sequence in which the adenosine residue repeats are essential for high-affinity recognition. An analysis of the expression profiles of the genes encoding the TcZFP2-associated mRNAs throughout the parasite life cycle by microarray hybridisation showed that most of the associated mRNAs were upregulated in the metacyclic trypomastigote forms, also suggesting a role for TcZFP2 in metacyclic trypomastigote differentiation. Knockdown of the orthologous Trypanosoma brucei protein levels showed ZFP2 to be a positive regulator of specific target mRNA abundance.