Release of plasma atrial natriuretic peptide after volume expansion is not related to pituitary-adrenal axis diurnal variation in normal subjects

The existence of a circadian rhythm of atrial natriuretic peptide (ANP) in humans is controversial. We studied the plasma ANP response to isotonic blood volume expansion in the morning and in the afternoon and its relationship with adrenocorticotropic hormone (ACTH)-cortisol diurnal variation in seven normal subjects. Basal plasma ANP level was similar in the morning (19.6 ± 2.4 pg/ml) and in the afternoon (21.8 ± 4.8 pg/ml). The ANP peak obtained with saline infusion (0.9% NaCl, 12 ml/kg) in the morning (49.4 ± 8 pg/ml) did not differ from that obtained in the afternoon (60.3 ± 10.1 pg/ml). There was no correlation between the individual mean cortisol and ACTH levels and the ANP peak obtained with saline infusion. These data indicate no diurnal variation in plasma ANP secretion induced by blood volume expansion and no relationship between plasma ANP peak and ACTH-cortisol diurnal variation

Circadian time-dependent effects of fencamfamine on inhibition of dopamine uptake and release in rat striatal slices

Fencamfamine (FCF) is a central stimulant that facilitates central dopaminergic transmission through inhibition of dopamine uptake and enhanced release of the transmitter. We evaluated the changes in the inhibition of uptake and the release of striatal [3H]-dopamine at 9:00 and 21:00 h, times corresponding to maximal and minimal behavioral responses to FCF, respectively. Adult male Wistar rats (200-250 g) maintained on a 12-h light/12-h dark cycle (lights on at 7:00 h) were used. In the behavioral experiments the rats (N = 8 for each group) received FCF (3.5 mg/kg, ip) or saline at 9:00 or 21:00 h. Fifteen minutes after treatment the duration of activity (sniffing, rearing and locomotion) was recorded for 120 min. The basal motor activity was higher (28.6 ± 4.2 vs 8.4 ± 3.5 s) after saline administration at 21:00 h than at 9:00 h. FCF at a single dose significantly enhanced the basal motor activity (38.3 ± 4.5 vs 8.4 ± 3.5 s) and increased the duration of exploratory activity (38.3 ± 4.5 vs 32.1 ± 4.6 s) during the light, but not the dark phase. Two other groups of rats (N = 6 for each group) were decapitated at 9:00 and 21:00 h and striata were dissected for dopamine uptake and release assays. The inhibition of uptake and release of [3H]-dopamine were higher at 9:00 than at 21:00 h, suggesting that uptake inhibition and the release properties of FCF undergo daily variation. These data suggest that the circadian time-dependent effects of FCF might be related to a higher susceptibility of dopamine presynaptic terminals to the action of FCF during the light phase which corresponds to the rats' resting period

Evidence of interaction between fluoxetine and isosorbide dinitrate on neuroleptic-induced catalepsy in mice

Drugs which influence 5-HTergic mechanisms can modify neuroleptic-induced catalepsy (NC) in rodents, a phenomenon produced by striatal dopamine (DA) receptor blockade. Previous research also suggests a role for endogenous nitric oxide (NO) in the modulation of striatal DAergic neurotransmission; in addition, NO seems to play a role in the 5-HT reuptake mechanism. It is known that clomipramine potentiates NC in mice, but the reported effects of selective 5-HT reuptake inhibitors (SSRIs) in this model are rather contradictory. We then decided to re-address this issue, investigating the effect of fluoxetine (FX), an SSRI, on NC. In view of the ubiquitous role of NO as a central neuromodulator, we also studied the effect of isosorbide dinitrate (ID), a centrally active NO donor, and how both drugs interact to affect the phenomenon of NC. Catalepsy was induced in male albino mice with haloperidol (H; 1 mg/kg, ip) and measured at 30-min interval by means of a bar test. Drugs (FX, ID and FX + ID) or saline (controls) were injected ip 30 min before H, with each animal used only once. FX (5 mg/kg) significantly reduced NC, with maximal attenuation (about 74%) occurring at 150 min after H. ID (5 mg/kg) also inhibited NC (150 min: 62% attenuation). The combined drugs (FX + ID group), however, caused a great potentiation of NC (4.7-fold at its maximum, at 90 min). The effect observed with ID is compatible with the hypothesis that NO increases DA release in the striatum. The attenuation of NC observed with FX may be due to a preferential net effect on the raphe somatodendritic synapse, where inhibitory 5-HT1A autoreceptors are operative. The enhancement of NC caused by combined administration of FX and ID suggests the presence of a pharmacodynamic interaction, whose mechanism, still unclear, may be related to a decrease in striatal DA release

Endothelium-dependent vasodilation in response to Pseudomonas aeruginosa lipopolysaccharide: an in vitro study on canine arteries

Early systemic arterial hypotension is a common clinical feature of Pseudomonas septicemia. To determine if Pseudomonas aeruginosa endotoxin induces the release of endothelium-derived nitric oxide (EDNO), an endogenous nitrovasodilator, segments of canine femoral, renal, hepatic, superior mesenteric, and left circumflex coronary arteries were suspended in organ chambers (physiological salt solution, 95% O2/5% CO2, pH 7.4, 37oC) to measure isometric force. In arterial segments contracted with 2 µM prostaglandin F2a, Pseudomonas endotoxin (lipopolysaccharide (LPS) serotype 10(Habs) from Pseudomonas aeruginosa (0.05 to 0.50 mg/ml)) induced concentration-dependent relaxation of segments with endothelium (P<0.05) but no significant change in tension of arteries without endothelium. Endothelium-dependent relaxation in response to Pseudomonas LPS occurred in the presence of 1 µM indomethacin, but could be blocked in the coronary artery with 10 µM NG-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of nitric oxide synthesis from L-arginine. The inhibitory effect of L-NMMA on LPS-mediated vasorelaxation of the coronary artery could be reversed by exogenous 100 µM L-arginine but not by 100 µM D-arginine. These experiments indicate that Pseudomonas endotoxin induces synthesis of nitric oxide from L-arginine by the vascular endothelium. LPS-mediated production of EDNO by the endothelium, possibly through the action of constitutive nitric oxide synthase (NOSc), may decrease systemic vascular resistance and may be the mechanism of early hypotension characteristic of Pseudomonas septicemia.

Purification and binding analysis of the nitrogen fixation regulatory NifA protein from Azospirillum brasilense

NifA protein activates transcription of nitrogen fixation operons by the alternative sigma54 holoenzyme form of RNA polymerase. This protein binds to a well-defined upstream activator sequence (UAS) located at the -200/-100 position of nif promoters with the consensus motif TGT-N10-ACA. NifA of Azospirillum brasilense was purified in the form of a glutathione-S-transferase (GST)-NifA fusion protein and proteolytic release of GST yielded inactive and partially soluble NifA. However, the purified NifA was able to induce the production of specific anti-A. brasilense NifA-antiserum that recognized NifA from A. brasilense but not from K. pneumoniae. Both GST-NifA and NifA expressed from the E. coli tac promoter are able to activate transcription from the nifHDK promoter but only in an A. brasilense background. In order to investigate the mechanism that regulates NifA binding capacity we have used E. coli total protein extracts expressing A. brasilense nifA in mobility shift assays. DNA fragments carrying the two overlapping, wild-type or mutated UAS motifs present in the nifH promoter region revealed a retarded band of related size. These data show that the binding activity present in the C-terminal domain of A. brasilense NifA protein is still functional even in the presence of oxygen.

Staircase in mammalian muscle without light chain phosphorylation

In disuse atrophied skeletal muscle, the staircase response is virtually absent and light chain phosphorylation does not occur. The purpose of the present study was to determine if staircase could be restored in atrophied muscle with continued absence of myosin light chain phosphorylation, by reducing what appears to be an otherwise enhanced calcium release. Control (untreated) and sham-operated female Sprague-Dawley rats were compared with animals after 2 weeks of complete inactivity induced by tetrodotoxin (TTX) application to the left sciatic nerve. In situ isometric contractile responses of rat gastrocnemius muscle were analyzed before and after administration of dantrolene sodium (DS), a drug which is known to inhibit Ca2+ release in skeletal muscle. Twitch active force (AF) was attenuated by DS from 2.2 ± 0.2 N, 2.7 ± 0.1 N and 2.4 ± 0.2 N to 0.77 ± 0.2 N, 1.05 ± 0.1 N and 1.01 ± 0.2 N in TTX (N = 5), sham (N = 11) and control (N = 7) muscles, respectively. Following dantrolene treatment, 10 s of 10-Hz stimulation increased AF to 1.32 ± 0.2 N, 1.52 ± 0.1 N and 1.45 ± 0.2 N for the TTX, sham and control groups, respectively, demonstrating a positive staircase response. Regulatory light chain (R-LC) phosphorylation was lower for TTX-treated (5.5 ± 5.5%) than for control (26.1 ± 5.3%) and sham (20.0 ± 5%) groups. There was no significant change from resting levels for any of the groups after DS treatment (P = 0.88). This study shows that treatment with dantrolene permits staircase in atrophied muscle as well as control muscle, by a mechanism which appears to be independent of R-LC phosphorylation.

Poly-DL-lactide-co-glycolide microspheres as a controlled release antigen delivery system

Successful vaccine application means maximum protection with minimal number of administrations. A rational development of vaccines involves studies of the nature of the antigen as well as of the adjuvant to be used to improve the immune responses. This has provided the impetus for studies to design the degradable devices and for different approaches to antigen delivery by different routes of administration. The development of controlled release systems based on polymeric devices that permit a sustained or pulsed release of encapsulated antigens has attracted much interest. Polymeric delivery systems consist of polymers that release their content continuously in a controlled manner over a period of time. The development of a biocompatible delivery system for parenteral administration offers several advantages in terms of immunoadjuvanticity over other compounds. It was found that, in contrast to other carriers, microspheres are more stable, thus permitting administration by the oral or parenteral route. In the present study, we describe the main characteristics and potentialities of this new immunoadjuvant for oral and parenteral administration.

Atropine and ODQ antagonize tetanic fade induced by L-arginine in cats

Although it has been demonstrated that nitric oxide (NO) released from sodium nitrite induces tetanic fade in the cat neuromuscular preparations, the effect of L-arginine on tetanic fade and its origin induced by NO have not been studied in these preparations. Furthermore, atropine reduces tetanic fade induced by several cholinergic and anticholinergic drugs in these preparations, whose mechanism is suggested to be mediated by the interaction of acetylcholine with inhibitory presynaptic muscarinic receptors. The present study was conducted in cats to determine the effects of L-arginine alone or after pretreatment with atropine or 1H-[1,2,4]oxadiazole [4,3-a]quinoxalin-1-one (ODQ) on neuromuscular preparations indirectly stimulated at high frequency. Drugs were injected into the middle genicular artery. L-arginine (2 mg/kg) and S-nitroso-N-acetylpenicillamine (SNAP; 16 µg/kg) induced tetanic fade. The Nw-nitro-L-arginine (L-NOARG; 2 mg/kg) alone did not produce any effect, but reduced the tetanic fade induced by L-arginine. D-arginine (2 mg/kg) did not induce changes in tetanic fade. The tetanic fade induced by L-arginine or SNAP was reduced by previous injection of atropine (1.0 µg/kg) or ODQ (15 µg/kg). ODQ alone did not change tetanic fade. The data suggest that the NO-synthase-GC pathway participates in the L-arginine-induced tetanic fade in cat neuromuscular preparations. The tetanic fade induced by L-arginine probably depends on the action of NO at the presynaptic level. NO may stimulate guanylate cyclase increasing acetylcholine release and thereby stimulating presynaptic muscarinic receptors.

Neuronal and endothelial nitric oxide synthase gene knockout mice

Targeted disruption of the neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS) genes has led to knockout mice that lack these isoforms. These animal models have been useful to study the roles of nitric oxide (NO) in physiologic processes. nNOS knockout mice have enlarged stomachs and defects in the inhibitory junction potential involved in gastrointestinal motility. eNOS knockout mice are hypertensive and lack endothelium-derived relaxing factor activity. When these animals are subjected to models of focal ischemia, the nNOS mutant mice develop smaller infarcts, consistent with a role for nNOS in neurotoxicity following cerebral ischemia. In contrast, eNOS mutant mice develop larger infarcts, and show a more pronounced hemodynamic effect of vascular occlusion. The knockout mice also show that nNOS and eNOS isoforms differentially modulate the release of neurotransmitters in various regions of the brain. eNOS knockout mice respond to vessel injury with greater neointimal proliferation, confirming that reduced NO levels seen in endothelial dysfunction change the vessel response to injury. Furthermore, eNOS mutant mice still show a protective effect of female gender, indicating that the mechanism of this protection cannot be limited to upregulation of eNOS expression. The eNOS mutant mice also prove that eNOS modulates the cardiac contractile response to ß-adrenergic agonists and baseline diastolic relaxation. Atrial natriuretic peptide, upregulated in the hearts of eNOS mutant mice, normalizes cGMP levels and restores normal diastolic relaxation.

How are antibodies involved in the protective mechanism of susceptible mice infected with T. cruzi?

Host resistance to Trypanosoma cruzi is dependent on both natural and acquired immune responses. During the acute phase of the infection the presence of IFN-g, TNF-a, IL-12 and GM-CSF has been closely associated with resistance, whereas TGF-ß and IL-10 have been associated with susceptibility. Several investigators have demonstrated that antibodies are responsible for the survival of susceptible animals in the initial phase of infection and for the maintenance of low levels of parasitemia in the chronic phase. However, how this occurs is not yet understood. Our results and other data in the literature support the hypothesis that the protective role of antibodies in the acute phase of infection is dependent mostly on their ability to induce removal of bloodstream trypomastigotes from the circulation in addition to other concomitant cell-mediated events.

Coexistence of potentiation and fatigue in skeletal muscle

Twitch potentiation and fatigue in skeletal muscle are two conditions in which force production is affected by the stimulation history. Twitch potentiation is the increase in the twitch active force observed after a tetanic contraction or during and following low-frequency stimulation. There is evidence that the mechanism responsible for potentiation is phosphorylation of the regulatory light chains of myosin, a Ca2+-dependent process. Fatigue is the force decrease observed after a period of repeated muscle stimulation. Fatigue has also been associated with a Ca2+-related mechanism: decreased peak Ca2+ concentration in the myoplasm is observed during fatigue. This decrease is probably due to an inhibition of Ca2+ release from the sarcoplasmic reticulum. Although potentiation and fatigue have opposing effects on force production in skeletal muscle, these two presumed mechanisms can coexist. When peak myoplasmic Ca2+ concentration is depressed, but myosin light chains are relatively phosphorylated, the force response can be attenuated, not different, or enhanced, relative to previous values. In circumstances where there is interaction between potentiation and fatigue, care must be taken in interpreting the contractile responses.

Oxytocin is a cardiovascular hormone

Oxytocin (OT), a nonapeptide, was the first hormone to have its biological activities established and chemical structure determined. It was believed that OT is released from hypothalamic nerve terminals of the posterior hypophysis into the circulation where it stimulates uterine contractions during parturition, and milk ejection during lactation. However, equivalent concentrations of OT were found in the male hypophysis, and similar stimuli of OT release were determined for both sexes, suggesting other physiological functions. Indeed, recent studies indicate that OT is involved in cognition, tolerance, adaptation and complex sexual and maternal behaviour, as well as in the regulation of cardiovascular functions. It has long been known that OT induces natriuresis and causes a fall in mean arterial pressure, both after acute and chronic treatment, but the mechanism was not clear. The discovery of the natriuretic family shed new light on this matter. Atrial natriuretic peptide (ANP), a potent natriuretic and vasorelaxant hormone, originally isolated from rat atria, has been found at other sites, including the brain. Blood volume expansion causes ANP release that is believed to be important in the induction of natriuresis and diuresis, which in turn act to reduce the increase in blood volume. Neurohypophysectomy totally abolishes the ANP response to volume expansion. This indicates that one of the major hypophyseal peptides is responsible for ANP release. The role of ANP in OT-induced natriuresis was evaluated, and we hypothesized that the cardio-renal effects of OT are mediated by the release of ANP from the heart. To support this hypothesis, we have demonstrated the presence and synthesis of OT receptors in all heart compartments and the vasculature. The functionality of these receptors has been established by the ability of OT to induce ANP release from perfused heart or atrial slices. Furthermore, we have shown that the heart and large vessels like the aorta and vena cava are sites of OT synthesis. Therefore, locally produced OT may have important regulatory functions within the heart and vascular beds. Such functions may include slowing down of the heart or the regulation of local vascular tone.

Angiotensin-(1-7) potentiates the coronary vasodilatatory effect of bradykinin in the isolated rat heart

It has been shown that angiotensin-(1-7) (Ang-(1-7)) infusion potentiates the bradykinin (BK)-induced hypotensive response in conscious rats. The present study was conducted to identify Ang-(1-7)-BK interactions in the isolated rat heart perfused according to the Langendorff technique. Hearts were excised and perfused through the aortic stump under a constant flow with Krebs-Ringer solution and the changes in perfusion pressure and heart contractile force were recorded. Bolus injections of BK (2.5, 5, 10 and 20 ng) produced a dose-dependent hypotensive effect. Ang-(1-7) added to the perfusion solution (2 ng/ml) did not change the perfusion pressure or the contractile force but doubled the hypotensive effect of the lower doses of BK. The BK-potentiating Ang-(1-7) activity was blocked by pretreatment with indomethacin (5 mg/kg, ip) or L-NAME (30 mg/kg, ip). The Ang-(1-7) antagonist A-779 (50 ng/ml in Krebs-Ringer) completely blocked the effect of Ang-(1-7) on BK-induced vasodilation. These data suggest that the potentiation of the BK-induced vasodilation by Ang-(1-7) can be attributed to the release of nitric oxide and vasodilator prostaglandins through an Ang-(1-7) receptor-mediated mechanism.

Treatment of cytomegalovirus retinitis with an intraocular sustained-release ganciclovir implant

The objective of this prospective study was to evaluate the efficacy and complications of the use of an intraocular sustained-release ganciclovir implant for the treatment of active cytomegalovirus (CMV) retinitis in AIDS patients. Thirty-nine eyes of 26 patients were submitted to ocular surgery. All patients underwent complete ocular examination before and after surgery. The surgical procedure was always done under local anesthesia using the same technique. The mean time for the surgical procedure was 20 min (range, 15 to 30 min). The average follow-up period was 3.7 months. Of all patient, only 4 presented recurrence of retinitis after 8, 8, 9 and 2 months, respectively. Three of them received a successful second implant. All 39 eyes of the 26 patients presented healing of retinitis as shown by clinical improvement evaluated by indirect binocular ophthalmoscopy and retinography. Retinitis healed within a period of 4 to 6 weeks in all patients, with clinical regression signs from the third week on. Six (15.4%) eyes developed retinal detachment. None of the patients developed CMV retinitis in the contralateral eye. The intraocular implant proved to be effective in controlling the progression of retinitis for a period of up to 8 months even in patients for whom systemic therapy with either ganciclovir or foscarnet or both had failed. The intraocular sustained-release ganciclovir implant proved to be a safe new procedure for the treatment of CMV retinitis, avoiding the systemic side effects caused by the intravenous medications and improving the quality of life of the patients.

A moderate decrease in temperature inhibits the calcium signaling mechanism(s) of the regulatory volume decrease in chick embryo cardiomyocytes

Chick cardiomyocytes, when submitted to hyposmotic swelling, exhibit a partial regulatory volume decrease (RVD). A Ca2+ influx by stretch-activated channels signals a taurine efflux and the RVD at 37°C. We evaluated the cell's performance at room temperature. Cardiomyocytes isolated and cultured from 11-day-old chick embryos were submitted to a hyposmotic solution (180 mOsm/kg H2O) at 37°C and at room temperature (26°C). Under these conditions we measured the changes in cell volume as well as the intracellular free Ca2+ (using fura-2). During hyposmotic swelling, cells at 37°C displayed a peak relative volume of 1.61 ± 0.03 and recovery to 1.22 ± 0.04 (N = 14), while cells at 26°C presented a peak swell relative volume of 1.74 ± 0.06 and did not recover (1.59 ± 0.09, N = 9). Transient increases in intracellular Ca2+, which are characteristic of the normal RVD, were observed at both temperatures (29.1 ± 4.5% (N = 8) and 115.2 ± 42.8% (N = 5) increase at 37° and 26°C (P<0.05), respectively). A delay in the Ca2+ transient increase was also observed when the cells were at 26°C (109 ± 34 s compared to 38 ± 9 s at 37°C, P<0.05). At room temperature the RVD does not occur because the calcium transient increase, which is an early event in the signaling of the RVD, is delayed. Also, free calcium is not cleared as in the 37°C RVD. In the normal RVD the free calcium returns to baseline levels. The very high and persistent free calcium levels seen at room temperature can lead to unregulated enzyme activities and may promote irreversible injury and cell death.